U.S. patent application number 10/279733 was filed with the patent office on 2003-10-16 for targeted thrombosis.
Invention is credited to Edgington, Thomas S., Liu, Cheng.
Application Number | 20030194400 10/279733 |
Document ID | / |
Family ID | 26990155 |
Filed Date | 2003-10-16 |
United States Patent
Application |
20030194400 |
Kind Code |
A1 |
Liu, Cheng ; et al. |
October 16, 2003 |
Targeted thrombosis
Abstract
The invention provided compositions and methods to initiate
site-specific thrombosis in tumor vasculature. The present
invention also provides methods for using the disclosed
compositions and methods to treat tumors.
Inventors: |
Liu, Cheng; (Carlsbad,
CA) ; Edgington, Thomas S.; (La Jolla, CA) |
Correspondence
Address: |
SCHWEGMAN, LUNDBERG, WOESSNER & KLUTH, P.A.
P.O. BOX 2938
MINNEAPOLIS
MN
55402
US
|
Family ID: |
26990155 |
Appl. No.: |
10/279733 |
Filed: |
October 24, 2002 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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60412194 |
Sep 20, 2002 |
|
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60336331 |
Oct 26, 2001 |
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Current U.S.
Class: |
424/94.64 ;
424/178.1; 424/450; 435/188.5; 435/226 |
Current CPC
Class: |
A61K 31/198 20130101;
C07K 14/745 20130101; A61K 31/704 20130101; A61K 31/704 20130101;
A61K 38/05 20130101; A61K 38/4846 20130101; A61K 31/198 20130101;
A61K 31/519 20130101; A61P 35/00 20180101; A61K 31/4245 20130101;
A61K 2300/00 20130101; A61K 45/06 20130101; C07K 2319/20 20130101;
A61K 9/127 20130101; A61K 31/4245 20130101; A61K 38/00 20130101;
A61K 2300/00 20130101; A61K 2300/00 20130101; A61K 2300/00
20130101; A61K 2300/00 20130101; A61K 31/519 20130101; C07K 2319/74
20130101; A61K 38/05 20130101; A61K 38/4846 20130101; A61K 2300/00
20130101 |
Class at
Publication: |
424/94.64 ;
424/178.1; 435/188.5; 435/226; 424/450 |
International
Class: |
A61K 038/48; A61K
039/395; A61K 009/127; C12N 009/64 |
Goverment Interests
[0002] This invention was made with Government support under Grant
No. PO1 HL16411 awarded by the National Institutes of Health. The
Government has certain rights in this invention.
Claims
What is claimed
1. A Selective Tissue Vascular Thrombogen comprising a Selective
Binding Domain associated with a Tissue Factor polypeptide, wherein
the Selective Binding Domain can bind to a channel for blood within
a tissue and the human tissue factor can initiate thrombosis within
the channel.
2. The Selective Tissue Vascular Thrombogen of claim 1 wherein the
tissue is a lung, breast, ovary, stomach, pancreas, larynx,
esophagus, testes, liver, parotid, biliary tract, colon, rectum,
cervix, uterus, endometrium, kidney, bladder, prostate, thyroid,
benign prostate hyperplasia, squamous cell carcinoma,
adenocarcinoma, small cell carcinoma, melanoma, glioma, or
neuroblastoma tumor.
3. The Selective Tissue Vascular Thrombogen of claim 1 wherein the
tissue is a prostate tumor.
4. The Selective Tissue Vascular Thrombogen of claim 1 wherein the
Tissue Factor polypeptide comprises SEQ ID NO: 3, SEQ ID NO: 4, SEQ
ID NO: 5 or SEQ ID NO: 6.
5. The Selective Tissue Vascular Thrombogen of claim 1 wherein the
Selective Binding Domain comprises a ligand for a cellular
receptor, a receptor for a cellular ligand or an inhibitor for a
membrane-associated protein.
6. The Selective Tissue Vascular Thrombogen of claim 1 wherein the
Selective Binding Domain binds selectively to endoglin, integrin,
VEGF receptor, or Prostate Specific Membrane Antigen.
7. The Selective Tissue Vascular Thrombogen of claim 1 wherein the
Selective Binding Domain is an integrin binding site from
fibronectin.
8. The Selective Tissue Vascular Thrombogen of claim 7 wherein the
integrin binding site from fibronectin is a polypeptide from SEQ ID
NO: 8.
9. The Selective Tissue Vascular Thrombogen of claim 1 wherein the
Selective Binding Domain is not an antibody.
10. The Selective Tissue Vascular Thrombogen of claim 1 wherein the
Selective Binding Domain comprises an inhibitor of prostate
specific membrane antigen.
11. The Selective Tissue Vascular Thrombogen of claim 10 wherein
the inhibitor of prostate specific membrane antigen comprises
Asp-.beta.-B-Glu, N-succinyl-glutamic acid, quisqalic acid or
2-(phosphonomethyl)pentanedioic acid.
12. The Selective Tissue Vascular Thrombogen of claim 1 comprising
SEQ ID NO: 9 or SEQ ID NO: 10.
13. A therapeutic composition comprising a therapeutically
effective amount of a Selective Tissue Vascular Thrombogen and a
pharmaceutically acceptable carrier, wherein the Selective Tissue
Vascular Thrombogen comprises a Selective Binding Domain associated
with a Tissue Factor polypeptide, wherein the Selective Binding
Domain can bind to a channel for blood within a tissue and the
Tissue Factor polypeptide can initiate thrombosis within the
channel.
14. The therapeutic composition of claim 13 wherein the tissue is a
lung, breast, ovary, stomach, pancreas, larynx, esophagus, testes,
liver, parotid, biliary tract, colon, rectum, cervix, uterus,
endometrium, kidney, bladder, prostate, thyroid, benign prostate
hyperplasia, squamous cell carcinoma, adenocarcinoma, small cell
carcinoma, melanoma, glioma, or neuroblastoma tumor.
15. The therapeutic composition of claim 13 wherein the tissue is a
prostate tumor.
16. The therapeutic composition of claim 13 wherein the Tissue
Factor polypeptide comprises SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO:
5 or SEQ ID NO: 6.
17. The therapeutic composition of claim 13 wherein the Selective
Binding Domain comprises a ligand for a cellular receptor, a
receptor for a cellular ligand or an inhibitor for a
membrane-associated protein.
18. The therapeutic composition of claim 13 wherein the Selective
Binding Domain binds selectively to endoglin, integrin, VEGF
receptor, or Prostate Specific Membrane Antigen.
19. The therapeutic composition of claim 13 wherein the Selective
Binding Domain is an integrin binding site from fibronectin.
20. The therapeutic composition of claim 19 wherein the integrin
binding site from fibronectin is a polypeptide from SEQ ID NO:
8.
21. The therapeutic composition of claim 13 wherein the Selective
Binding Domain is not an antibody.
22. The therapeutic composition of claim 13 wherein the Selective
Binding Domain comprises an inhibitor of prostate specific membrane
antigen.
23. The therapeutic composition of claim 22 wherein the inhibitor
of prostate specific membrane antigen comprises Asp-.beta.-Glu,
N-succinyl-glutamic acid, quisqalic acid, or
2-(phosphonomethyl)pentanedi- oic acid.
24. The therapeutic composition of claim 13 wherein the
pharmaceutically acceptable carrier is a liposome.
25. The therapeutic composition of claim 13 that further comprises
a Factor VII or Factor VIIa polypeptide.
26. The therapeutic composition of claim 13 that further comprises
a chemotherapeutic agent.
27. The therapeutic composition of claim 13 wherein the Selective
Tissue Vascular Thrombogen comprises SEQ ID NO: 9 or SEQ ID NO:
10.
28. A method of treating a solid tumor in an animal that comprises
administering a therapeutically effective amount of a Selective
Tissue Vascular Thrombogen comprising a Selective Binding Domain
associated with a Tissue Factor polypeptide, wherein the Selective
Binding Domain can bind to a channel for blood within a tumor and
the Tissue Factor polypeptide can initiate thrombosis within the
channel.
29. The method of claim 28 wherein the tumor is a lung, breast,
ovary, stomach, pancreas, larynx, esophagus, testes, liver,
parotid, biliary tract, colon, rectum, cervix, uterus, endometrium,
kidney, bladder, prostate, thyroid, squamous cell carcinoma,
adenocarcinoma, small cell carcinoma, melanoma, glioma, or
neuroblastoma tumor.
30. The method of claim 28 wherein the tumor is a prostate
tumor.
31. The method of claim 30 wherein the thrombosis leads to tumor
necrosis.
32. The method of claim 28 wherein the tissue factor polypeptide
comprises SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5 or SEQ ID NO:
6.
33. The method of claim 28 wherein the Selective Binding Domain
comprises a ligand for a cellular receptor, a receptor for a
cellular ligand or an inhibitor for a membrane-associated
protein.
34. The method of claim 28 wherein the Selective Binding Domain
binds selectively to endoglin, integrin, VEGF receptor, or Prostate
Specific Membrane Antigen.
35. The method of claim 28 wherein the Selective Binding Domain is
an integrin binding site from fibronectin.
36. The method of claim 35 wherein the integrin binding site from
fibronectin is a polypeptide from SEQ ID NO: 8.
37. The method of claim 28 wherein the Selective Binding Domain is
not an antibody.
38. The method of claim 28 wherein the Selective Binding Domain
comprises an inhibitor of prostate specific membrane antigen.
39. The method of claim 38 wherein the inhibitor of prostate
specific membrane antigen comprises Asp-.beta.-Glu,
N-succinyl-glutamic acid, quisqalic acid or
2-(phosphonomethyl)pentanedioic acid.
40. The method of claim 28 wherein the Selective Tissue Vascular
Thrombogen is administering in a liposome.
41. The method of claim 28 that further comprises administering a
therapeutically effective amount of a chemotherapeutic agent.
42. The method of claim 41 wherein the chemotherapeutic agent
comprises methotrexate or doxorubicin.
43. The method of claim 28 that further comprises administering a
therapeutically effective amount of an inhibitor of prostate
specific membrane antigen.
44. The method of claim 43 wherein the inhibitor of prostate
specific membrane antigen comprises Asp-.beta.-Glu,
N-succinyl-glutamic acid, quisqalic acid or
2-(phosphonomethyl)pentanedioic acid.
45. The method of claim 28 wherein the Selective Tissue Vascular
Thrombogen comprises SEQ ID NO: 9 or SEQ ID NO: 10.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application is a non-provisional application which
claims priority to provisional application entitled "Targeted
Thrombosis" filed Sep. 20, 2002, U.S. Serial No. 60/412,194 and
provisional application entitled "Targeted Thrombosis" filed Oct.
26, 2001, U.S. Serial No. 60/336,331, the specifications of which
are incorporated herein by reference.
FIELD OF THE INVENTION
[0003] The present invention relates to the fields of blood
coagulation, thrombosis, tumor angiogenesis, and cancer therapy.
The present invention provides various compositions and methods to
treat solid tumors by inducing site-selective thrombosis in tumor
blood vessels.
BACKGROUND OF THE INVENTION
[0004] Although many advances in cancer therapy have been made
during the last thirty years, many prevalent forms of human cancer
currently resist chemotherapeutic intervention. For example,
prostate cancer is the second leading cause of cancer death in men.
The incidence of prostate cancer has increased 141.8% between 1973
and 1994. In 1998, new prostate cancer cases totaled 184,500,
representing about one new case every three minutes, and 29% of all
new cancer cases in American men. In 1998, an estimated 39,200 men
died of prostate cancer. A life is lost to prostate cancer in this
country every 13 minutes. According to the National Cancer
Institute, the annual cost of prostate cancer to the country,
including medical care, lost wages and lost productivity, may be as
high as $15 billion.
[0005] Certain types of tumors are more amenable to therapy than
others because they are more accessible to therapeutic agents. For
example, soft tissue tumors such as lymphomas, and tumors of the
blood and blood-forming organs such as leukemia, have generally
been more responsive to chemotherapeutic therapy than have solid
tumors such as carcinomas. One reason for the susceptibility of
soft and blood-based tumors to chemotherapy is that they are
physically more accessible to chemotherapeutic intervention. It is
simply more difficult for most chemotherapeutic agents to reach all
of the cells of a solid tumor mass than it is for such agents to
reach the cells of soft tumors and blood-based tumors. While it is
possible to increase dosages, chemotherapeutic agents are toxic at
higher doses. Hence, conventional anti-tumor agents generally have
a limited range of effectiveness against solid tumors and a need
exists for the development of novel strategies for the treatment of
solid tumors.
[0006] One strategy for treating solid tumors is to use anti-tumor
cell antibodies to deliver a toxin to the tumor cells. However,
this method suffers from certain drawbacks. For example,
antigen-negative or antigen-deficient cells can survive to
repopulate the tumor or lead to further metastasis. Also, a solid
tumor is generally impermeable to large molecules like antibodies,
especially when linked to a toxin molecule.
[0007] Recently, there is increasing interest in developing methods
to induce site-selective thrombosis within blood vessels of a
selected tissue and thereby infarct and destroy that tissue. This
approach derived from the notion that in order for a tumor to grow
beyond a critical size, it must recruit and activate endothelial
cells to form its own new microvasculature (Denekamp 1990; Folkman
1992). Some investigators have therefore targeted tumor blood
vessels for destruction in order to destroy the supply of oxygen
and nutrients needed for local tumor cells to proliferate and
survive (Huang, Molema et al. 1997).
[0008] WO 96/01653 discloses antibodies against tumor vasculature
markers to deliver thrombogens to the vasculature of solid tumors.
Vascular targeting strategies are also described in Burrows et al.
(1992), in Burrows and Thorpe (1993) and in WO 93/17715. U.S. Pat.
No. 6,156,321 discloses that a truncated form of Tissue Factor can
bind to A20 lymphoma cells when co-administered with a bispecific
non-neutralizing antibody that binds to Tissue Factor and to an
antigen on the A20 lymphoma cells.
SUMMARY OF THE INVENTION
[0009] The invention provides Selective Tissue Vascular Thrombogens
(STVTs) that can induce targeted thrombosis, infarction and
destruction of selected tissues, for example, tumors. Targeting the
blood vessels of tumors has certain advantages in that it is not
likely to lead to the development of resistant tumor cells or
populations thereof. Delivery of Selective Tissue Vascular
Thrombogens to blood vessels avoids the accessibility problems
associated with targeting cells that are deep within a solid tumor.
Moreover, destruction of the blood vessels may have an amplified
anti-tumor effect because many tumor cells rely on a single vessel
for their oxygen and nutrient supply.
[0010] The Selective Tissue Vascular Thrombogens (STVTs) of the
invention are novel proteins having at least two functional
domains. The first functional domain is a Tissue Factor polypeptide
that can induce thrombogenesis, for example, the extracellular
domain of Tissue Factor. The second functional domain is a
Selective Binding Domain that can selectively bind to a
cell-specific or tissue-specific molecule. Preferably, the
Selective Binding Domain can bind to a molecule within a tumor, for
example, a molecule on the luminal surface of a tumor blood
channel. Upon binding, the Tissue Factor polypeptide can induce
thrombosis.
[0011] Additional domains may be incorporated into the Selective
Tissue Vascular Thrombogens of the invention. For example,
Selective Tissue Vascular Thrombogens can include membrane
associating domains or transmembrane domains of any protein known
to one of skill in the art. Other domains that can be incorporated
into the Selective Tissue Vascular Thrombogens of the invention
include spacer domains to optimize spacing and/or interaction or
non-interaction between elements of the domains.
[0012] Selective Binding Domains selectively localize the Selective
Tissue Vascular Thrombogens, for example, a thrombogenic Tissue
Factor domain, to a particular cell type, a particular tissue or a
particular tumor type. However, to efficiently induce thrombosis,
the Selective Binding Domain is selected to bind to a component
within a blood channel. More than one Selective Binding Domain can
be incorporated into the Selective Tissue Vascular Thrombogens of
the invention, for example, to enhance thrombogenic function, and
to increase the selectivity of localization or the selectivity of
action.
[0013] According to the invention, certain tumor cells can form
channels that mimic the function of blood vessels. The channels
formed by such tumor cells are deep within the solid tumor and join
with the normal circulatory system of the animal at the periphery
of the tumor. Hence, Selective Binding Domains are preferably tumor
cell membrane proteins that allow the Selective Tissue Vascular
Thrombogens of the invention to bind with specificity to a selected
tumor cell type.
[0014] Accordingly, the invention provides a Selective Tissue
Vascular Thrombogen comprising a Selective Binding Domain
associated with a Tissue Factor polypeptide. The Selective Binding
Domain can bind to a channel for blood within a tissue and the
human tissue factor can initiate thrombosis within the channel. The
Selective Tissue Vascular Thrombogen can be made by covalent or
non-covalent association of the Tissue Factor polypeptide with the
Selective Binding Domain.
[0015] Such Selective Tissue Vascular Thrombogens can bind to
channels within any tissue, for example, within a solid tumor. Such
tissues can be lung, breast, ovary, stomach, pancreas, larynx,
esophagus, testes, liver, parotid, biliary tract, colon, rectum,
cervix, uterus, endometrium, kidney, bladder, prostate, thyroid,
benign prostate hyperplasia, squamous cell carcinoma,
adenocarcinoma, small cell carcinoma, melanoma, glioma, or
neuroblastoma tissues or tumors. In one embodiment, the tissue is a
prostate tumor. The Tissue Factor polypeptide is preferably a human
Tissue Factor polypeptide, for example, a polypeptide comprising
SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5 or SEQ ID NO: 6.
[0016] The Selective Binding Domain can be any molecule, peptide or
polypeptide that can selectively bind or associate with a selected
cell or tissue type. For example, the Selective Binding Domain can
be a ligand for a cellular receptor, a receptor for a cellular
ligand, or an inhibitor for a membrane-associated protein. The
selective binding domain can, for example, bind selectively to
endoglin, integrin, VEGF receptor, a glycosaminoglycan or Prostate
Specific Membrane Antigen. In one embodiment, the selective binding
domain is an integrin binding site from fibronectin. In another
embodiment, the selective binding domain is an inhibitor of
prostate specific membrane antigen, for example, Asp-.beta.-Glu,
N-succinyl-glutamic acid or quisqalic acid.
[0017] In certain embodiments, the Selective Tissue Vascular
Thrombogen has SEQ ID NO: 9 or SEQ ID NO: 10.
[0018] The invention also provides therapeutic compositions
comprising a therapeutically effective amount of at least one
Selective Tissue Vascular Thrombogen of the invention and a
pharmaceutically acceptable carrier, wherein the Selective Tissue
Vascular Thrombogen comprises a Selective Binding Domain
associated, fused or attached to a Tissue Factor polypeptide,
wherein the Selective Binding Domain can bind to a channel for
blood within a tissue and the Tissue Factor polypeptide can
initiate a coagulation protease cascade within the channel. The
Selective Tissue Vascular Thrombogen composition can also include a
chemotherapeutic agent, a Factor VII polypeptide or a Factor VIIa
polypeptide. Liposomes are one example of pharmaceutically
acceptable carrier for the present compositions. In general, the
therapeutic compositions are administered intravenously.
[0019] The invention further provides a method of treating a solid
tumor in an animal that comprises administering a therapeutically
effective amount of a Selective Tissue Vascular Thrombogen
comprising a Selective Binding Domain fused or attached to a Tissue
Factor polypeptide, wherein the Selective Binding Domain can bind
to a channel for blood within a tumor and the human tissue factor
can induce coagulation within the channel. The compositions of the
invention can also be used in such therapeutic methods.
DESCRIPTION OF THE FIGURES
[0020] FIG. 1A provides a structural model of the ternary complex
of Tissue Factor (blue molecule in the middle that projects through
the cell membrane) with Factor VIIa (red molecule to the left) and
Factor X (yellow molecule to the right that extends down to become)
associated with a cell surface. The transmembrane domain of native
Tissue Factor spans the cell membrane and ensures proper
positioning of both Factor VIIa and Factor X (or Factor IX) on the
cell surface. The interaction of the N-terminal Gla domain of both
Factor VIIa and Factor X with the cell membrane is critical for the
full thrombogenic activity of this complex.
[0021] FIG. 1B shows a structural model of a ternary complex of a
Tissue-Selective Vascular Thrombogen of the invention where the
Tissue Factor polypeptide (in the middle) is associated with Factor
VIIa (on the left) and Factor X (on the right). The N-terminal
extracellular domain of Tissue Factor is fused with a Selective
Binding Domain (arrow in the upper left comer of the right panel
(FIG. 1B)) to form a novel Tissue-Selective Vascular Thrombogen.
The extreme portion of the N-terminus of Tissue Factor is not
involved in its function. Hence, addition to the N-terminus of a
Tissue Factor polypeptide of another molecule or domain is
possible. In this invention, a Selective Binding Domain can be
added to properly associate and physically align the Tissue Factor
polypeptide with the cell surface. Attachment of such a Selective
Binding Domain to its N-terminus does not adversely affect the
conformation or the function of the selected Tissue Factor
polypeptide. Factor VII, or its activated form, Factor VIIa, and
Factor X, or Factor IX, can therefore interact with the Tissue
Factor domain when aligned on a functionally supportive region of
an anionic cell surface. Such interaction permits formation of a
complex that has a conformation very similar to the structure of a
native Tissue Factor:Factor VIIa:Factor Xa complex. The
thrombogenic activity of this complex is substantially unaffected
by the incorporated Selective Binding Domain.
[0022] FIG. 2A is a silver-stained SDS polyacrylamide gel of an
electrophoretically purified fibronectin-Tissue Factor (Fn-TF)
construct (lane 1) and a polypeptide encoding the extracellular
domain of Tissue Factor (TF1-218) (lane 2). Only a single band was
observed for each sample, indicative of the homogeneity and the
purity of each protein preparation.
[0023] FIG. 2B is an anti-Tissue Factor western blot analysis of a
replica gel of FIG. 2A, in which purified Fn-TF and TF1-218
proteins are present in lanes 1 and lane 2, respectively.
[0024] FIG. 3 graphically illustrates the ability of a
fibronectin-Tissue Factor (Fn-TF) Tissue-Selective Vascular
Thrombogen to act as cofactor for enhancement of Factor VIIa
amidolytic activity. The amidolytic activity of Factor VIIa was
measured as a function of the concentration of Tissue Factor
(1-218) (.cndot.) or of the Fn-TF protein (O). These results
indicate that the affinity of the Tissue Factor domain for Factor
VIIa is not adversely affected by the incorporation of the
fibronectin domain. Accordingly, the subtle protein-protein
interactions between the Tissue Factor domain and the protease
domain of Factor VIIa that are responsible for allosteric induction
of Factor VIIa amidolytic function are not adversely affected.
[0025] FIG. 4 graphically illustrates the proteolytic activity of a
complex between the Fn-TF protein and Factor VIIa (O), as compared
with a complex between a Tissue Factor extracellular polypeptide
(TF 1-218) and Factor VIIa (.cndot.). Increased proteolytic
activity is observed with increasing concentrations of both Fn-TF
and Tissue Factor. The activity curves for the two are very
similar, suggesting that the incorporated fibronectin domain does
not affect the recognition of factor X by the Fn-TF:VIIa protease
complex.
[0026] FIG. 5 graphically illustrates the binding of the Fn-TF
protein to integrin expressing CHO K1 cells. The amount of Fn-TF
(O) bound increases as the Fn-TF concentration increases. In
contrast, soluble Tissue Factor (TF1-218)(.cndot.) shows no
appreciable association with CHO K1 cells.
[0027] FIG. 6 graphically illustrates the initiation of localized
coagulation on the cell surface by Fn-TF (O) or soluble Tissue
Factor (TF1-218) (.cndot.) using cells that express integrin (CHO
K1 cells). Coagulation time decreased with increasing
concentrations of Fn-TF but not with soluble TF. These data
indicate that the binding of Fn-TF to integrin led to the
regeneration of thrombogenic function by association of the Fn-TF
molecule with the cell surface through interaction with integrin.
These data also indicate that the Fn-TF protein can assume a
conformation that is substantially similar to that of native,
transmembranic Tissue Factor so that initiation of the coagulation
protease cascade is substantially unaffected by a heterologous
Selective Binding Domain.
[0028] FIG. 7 graphically compares the thrombogenic activity of the
Fn-TF protein with native Tissue Factor and further illustrates
that an RGDS (SEQ ID NO: 17) peptide can competitively inhibit
Fn-TF activity. The coagulation activity of the Fn-TF construct can
be almost completely inhibited with the RGDS peptide (Fn-TF (20
nM)+RGDS (500 .mu.M)). In the presence of RGDS, Fn-TF has low
activity similar to soluble Tissue Factor (sTF), which cannot
assemble into a thrombogenic complex on the cell. The RGDS peptide
can therefore compete for binding to integrin, thereby blocking the
binding of the fibronectin domain of Fn-TF to integrin. These data
further confirm that the coagulation activity of the Fn-TF
construct is dependent on the binding of a Selective Binding
Domain.
[0029] FIG. 8A is a photomicrograph of a formalin fixed section of
a LuCap 58 prostate tumor stained with the biotinylated anti-PSMA
antibody 7E11C-5. Note the intense positive stain of PSMA (arrows)
on the endothelial surface of the tumor microvasculature.
[0030] FIG. 8B is an expanded view of the inset identified on a
photomicrograph of FIG. 8A. Channels stained with biotinylated
anti-PSMA antibodies are identified (arrows). Note that the
intensity of the staining is greatest in the lining of the lumen
for each channel structure.
[0031] FIG. 8C is a photomicrograph showing LuCap xenograph tumor
cells stained red (arrows) with anti-PSMA antibody while most of
the surrounding endothelial cells stained green with anti-CD31
antibody. The red and green staining patterns are mutually
exclusive indicating that PSMA is not expressed on CD31 positive
endothelial cells.
[0032] FIG. 8D depicts a section of a rat Mat Lu tumor where PSMA
was observed to co-localize with cells that line the channels in
these tumors. Rats were injected intravenously with 10.sup.12 M 13
bacteriophage and tumors were harvested about 2 min. later. The
tumor channels stained red with anti-PSMA (J591) antibodies (arrow)
and the circulated phage were stained green with anti-phage
antibodies (arrows). These results indicate that the PSMA positive
cell lined channel structure is connected with the circulatory
system of the animal.
[0033] Together, FIGS. 8A-8D indicate that PSMA-positive cells are
tumor cells undergoing vasculogenic mimicry and forming channels
that constitute part of the tumor vasculature. FIG. 8E is a
schematic representation of such vasculogenic mimicry in which
tumor cells differentiate into an endothelial-like phenotype and
form channel structures that connect to natural endothelial-lined
tumor vessels and carry blood into the tumor.
[0034] FIG. 9A a silver-stained gel of purified Streptavidin-Tissue
Factor.
[0035] FIG. 9B illustrates the activity of the
D-.beta.-E-biotin:streptavi- din-Tissue Factor complex in a Factor
X generation assay. The D-.beta.-E-biotin:streptavidin-Tissue
Factor construct (filled circle) has much more activity than the
streptavidin-Tissue Factor construct (B symbols) that lacks the
D-.beta.-E Selective Binding Domain.
[0036] FIG. 10 provides a pathological analysis of Mat Lu tumor
treated with the D-.beta.-E-streptavidin-Tissue Factor protein.
[0037] FIG. 10A illustrates that the treated tumor (left) was
extensively necrotic compared to an untreated tumor (right). The
center of the treated tumor was liquefied. However, there was still
a rim of surviving tumor cells in the treated tumor.
[0038] FIG. 10B is a photomicrograph of a section of an untreated
Mat Lu tumor. The tumor is undifferentiated and the majority of the
tumor blood vessels are not visible.
[0039] FIG. 10C is a photomicrograph of a section of a Mat Lu tumor
after repeated treatment with the D-.beta.-E-streptavidin-Tissue
Factor protein. The center of the tumor is necrotic and has
collapsed into amorphous debris. Extensive vessel occlusion is
visible.
[0040] FIG. 10D is a photomicrograph of a thrombotic vessel
containing occlusive platelet aggregates, packed red blood cells,
and fibrin. There are also large numbers of inflammatory cells that
have infiltrated into the tumor.
[0041] FIG. 11A graphically depicts retardation of Mat Lu tumor
growth by the PSMA STVT targeting thrombogen. In the saline treated
control group (square symbols), the tumor volume increased
progressively and was greater than the
D.beta.E-biotin:streptavidin-TF:Factor VIIa treated group (J
symbols). The tumor size was measured with a caliper and tumor
volume calculated as D.times.d.sup.2. In this case, although tumor
center is necrotic and liquified, the total tumor size remained
unchanged from day zero or increased slightly as the surviving
tumor cells at the rim of the tumor continued to grow.
[0042] FIG. 11B graphically illustrates the weight of tumors after
dissection. The average tumor weight in the treated group (STVT,
white) was less than that of the control group (cross-hatched).
[0043] FIG. 12A illustrates that combined treatment with low doses
of liposomal doxorubicin and the D-.beta.-E-streptavidin-Tissue
Factor STVT augments the tumoricidal effect of PSMA directed STVT
therapy. In representative experiments, the combination of
doxorubicin and the STVT resulted in nearly complete growth arrest
of tumors in the treated animals (closed circles, n=12), in
striking contrast to those treated only with low dose liposomal
doxorubicin (closed squares, n=12). The data points represent mean
?SEM of 12 rats (p<0.001). The experiment was reproducible with
comparable results.
[0044] FIG. 12B graphically illustrates the percent survival of
animals treated with the STVT thrombogen or doxorubicin. As
illustrated, the combination of STVT thrombogen and doxorubicin
treatment (Dox+STVT, long dashed line - -) lead to significantly
better survival than mock-treated (thin solid line) animals.
Animals treated with the combination of STVT thrombogen and
doxorubicin (- -) also survived significantly longer than animals
treated with doxorubicin only (thick solid line) or with only the
STVT (short dashed line - - - )
[0045] FIG. 13A graphically illustrates that as the concentration
of D.beta.E inhibitor increases the viability of PSMA expressing
prostate cancer cells in culture declines. A cell proliferation and
viability assay was employed to assess D.beta.E inhibitor activity
using trypan blue staining. LnCap cells (4.times.10.sup.4
cells/well) were seeded in 96 well plates. Different concentrations
of the D.beta.E inhibitor or the Asp-Glu (D-E) substrate were added
to the media as indicated. The % cell viability was determined 48
hours after treatment as the number of living cells (unstained)
divided by total cells count (stain+unstained cells). Inhibition of
the glutamyl preferring carboxypeptidase activity of PSMA using its
inhibitor Aspartyl-.beta.-linked L glutamate (D-.beta.-E) resulted
in tumor cell death in a dose dependent manner in contrast to its
physiological substrate analogue, Aspartyl-glutamate (D-E).
[0046] FIG. 13B graphically illustrates the synergistic effect of
combining methotrexate (MTX) and the PSMA inhibitor D-.beta.-E
(filled circles), on cancer cell viability in vitro. The cytotoxic
effect of methotrexate was assessed with and without the presence
of the PSMA inhibitor (D-.beta.-E) or PSMA substrate (D-E) using a
cell proliferation and viability assay. Tumor cell viability was
less when cells were exposed to a combination of methotrexate and
the D-.beta.-E inhibitor (filled circles) than when cells were
exposed to methotrexate alone (filled triangles) or a combination
of methotrexate and the D-E substrate (open squares). The cytotoxic
effect of methotrexate was potentiated in the presence of inhibitor
at a concentration of 0.1 uM. The ID.sub.50 of MTX was reduced from
around 10 uM to around 0.5 uM in the presence of the PSMA inhibitor
(D-.beta.-E) (ID.sub.50/ID.sub.50*=20), a twenty-fold enhancement
of the tumoricidal activity.
DETAILED DESCRIPTION OF THE INVENTION
[0047] The present invention provides new compositions and methods
for targeted thrombosis at selected vascular sites within an
animal, for example, within tumors. Such targeted thrombosis is
achieved by administering novel Tissue-Selective Vascular
Thrombogens and compositions thereof. Such Tissue-Selective
Vascular Thrombogens contain at least two domains. The first domain
comprises a coagulation-activating Tissue Factor polypeptide. The
second domain is a Selective Binding Domain. The Selective Binding
Domain can recognize and bind to a selected cell type, for example,
a specific tumor cell type. More than one Tissue Factor polypeptide
and/or more than one Selective Binding Domain can be included in
the Tissue-Selective Vascular Thrombogens of the invention.
[0048] Other domains can be incorporated into the Tissue-Selective
Vascular Thrombogens of the invention. Such additional domains can
be used, for example, to help spatially orient one or more of the
other domains, to add additional Selective Binding Domains, to
facilitate insertion of the Tissue-Selective Vascular Thrombogen
into a cell membrane, to orient the Tissue-Selective Vascular
Thrombogen with the cell surface or to enhance, or prevent
neutralization of, the activity of the Tissue-Selective Vascular
Thrombogen.
[0049] These compositions and methods can be used to activate the
thrombogenic cascade in the tumor blood vessels, thereby blocking
blood flow to the tumor and killing tumor cells within the tumor.
The present invention provides that such compositions may be
administered alone, in combination with conventional
chemotherapeutics, in combination with Factor VIIa or other factors
involved in the cascade of events leading to localized
thrombogenesis.
[0050] Target Diseases
[0051] Angiogenesis in undesired locations is involved in wide
range of diseases. The concepts, compositions and methods provided
by this invention are broadly applicable to the treatment of any
disease that has a vascular component, including benign or
malignant tumors. Such vasculature-associated diseases include
benign prostate hyperplasia (BPH), diabetic retinopathy, vascular
restenosis, arteriovenous malformations (AVM), meningioma,
hemangioma, neovascular glaucoma and psoriasis; and also
angiofibroma, arthritis, atherosclerotic plaques, corneal graft
neovascularization, hemophilic joints, hypertrophic scars,
osler-weber syndrome, pyogenic granuloma retrolental fibroplasia,
scleroderma, trachoma, vascular adhesions, synovitis, dermatitis
and even endometriosis.
[0052] An important application of the present compositions and
methods is to treat solid tumors. Typical vascularized tumors are
the solid tumors, particularly carcinomas, which require a vascular
component for the provision of oxygen and nutrients via the blood.
Exemplary solid tumors that may be treated using the invention
include, but are not limited to, carcinomas of the lung, breast,
ovary, stomach, pancreas, larynx, esophagus, testes, liver,
parotid, biliary tract, colon, rectum, cervix, uterus, endometrium,
kidney, bladder, prostate, thyroid, squamous cell carcinomas,
adenocarcinomas, small cell carcinomas, melanomas, gliomas,
neuroblastomas, and the like.
[0053] Table 1 is provided for the purpose of exemplifying human
tumor cell lines that are publicly available. The information
presented in Table 1 is provides by means of an example, and not
intended to be limiting either by year or by scope. One of skill in
the art may consult the ATCC Catalogue of any subsequent year to
identify other appropriate cell lines. Also, if a particular cell
type is desired, the means for obtaining such cells, and/or their
instantly available source, will be known to those of skill in the
particular art. An analysis of the scientific literature can thus
readily reveal an appropriate choice of cell for any tumor cell
type to be targeted.
1TABLE 1 HUMAN TUMOR CELL LINES AND SOURCES ATTC/HTB CELL NUMBER
LINE TUMOR TYPE 1 J82 Transitional-cell carcinoma, bladder 2 RT4
Transitional-cell papilioma, bladder 3 ScaBER Squamous carcinoma,
bladder 4 T24 Transitional-cell carcinoma, bladder 5 TCCSUP
Transitional-cell carcinoma, bladder, primary grade IV 9 5637
Carcinoma, bladder, primary 10 SK-N-MC Neuroblastoma, metastasis to
supra-orbital area 11 SK-N-SH Neuroblastoma, metastasis to bone
marrow 12 SW 1088 Astrocytoma 13 SW 1783 Astrocytoma 14 U-87 MG
Glioblastoma, astrocytoma, grade III 15 U-118 MG Glioblastoma 16
U-138 MG Glioblastoma 17 U-373 MG Glioblastoma, astrocytoma, grade
III 18 Y79 Retinoblastoma 19 BT-20 Carcinoma, breast 20 BT-474
Ductal carcinoma, breast 22 MCF7 Breast adenocarcinoma, pleural
effusion 23 MDA-MB-134-VI Breast, ductal carcinoma, pleural
effusion 24 MDA-MU-157 Breast medulla, carcinoma, pleural effusion
25 MDA-MB-175-VII Breast, ductal carcinoma, pleural effusion 27
MDA-MB-361 Adenocarcinoma, breast, metastasis to brain 30 SK-BR-3
Adenocarcinoma, breast, malignant pleural effusion 31 C-33 A
Carcinoma, cervix 32 HT-3 Carcinoma, cervix, metastasis to lymph
node 33 ME-180 Epidermoid carcinoma, cervix, metastasis to omentum
34 MS751 Epidermoid carcinoma, cervix, metastasis to lymph node 35
SiHa Squamous carcinoma, cervix 36 JEG-3 Choriocarcinoma 37 Caco-2
Adenocarcinoma, colon 38 HT-29 Adenocarcinoma, colon, moderately
well-differentiated grade II 39 SK-CO-1 Adenocarcinoma, colon,
ascites 40 HuTu 80 Adenocarcinoma, duodenum 41 A-253 Epidermoid
carcinoma, submaxillary gland 43 FaDu Squamous cell carcinoma,
pharynx 44 A-498 Carcinoma, kidney 45 A-704 Adenocarcinoma, kidney
46 Caki-1 Clear cell carcinoma, consistent with renal primary, skin
metastasis 47 Caki-2 Clear cell carcinoma, consistent with renal
primary 48 SK-HEP-1 Wilms' tumor, pleural effusion 49 Sw 839
Adenocarcinoma, kidney 52 SK-HEP-1 Adenocarcinoma, liver, ascites
53 A-427 Carcinoma, lung 54 Calu-1 Epidermoid carcinoma grade III,
lung, metastasis to pleura 55 Calu-3 Adenocarcinoma, lung, pleural
effusion 56 Calu-6 Anaplastic carcinoma, probably lung 57 SK-LU-1
Adenocarcinoma, lung consistent with poorly differentiated, grade
III 58 SK-MES-1 Squamous carcinoma, lung, pleural effusion 59 SW
900 Squamous cell carcinoma, lung 60 EB1 Burkitt lymphoma, upper
maxilla 61 EB2 Burkitt lymphoma, ovary 62 P3HR-1 Burkitt lymphoma,
ascites 63 HT-144 Malignant melanoma, metastasis to subcutaneous
tissue 64 Malme-3M Malignant melanoma, metastasis to lung 66
RPMI-7951 Malignant melanoma, metastasis to lymph node 67 SK-MEL-1
Malignant melanoma, metastasis to lymphatic system 68 SK-MEL-2
Malignant melanoma, metastasis to skin of thigh 69 SK-MEL-3
Malignant melanoma, metastasis to lymph node 70 SK-MEL-S Malignant
melanoma, metastasis to auxiliary node 71 SK-MEL-24 Malignant
melanoma, metastasis to node 72 SK-MEL-28 Malignant melanoma 73
SK-MEL-31 Malignant melanoma 75 Caov-3 Adenocarcinoma, ovary,
consistent with primary 76 Caov-4 Adenocarcinoma, ovary, metastasis
to subserosa of fallopian tube 77 SK-OV-3 Adenocarcinoma, ovary,
malignant ascites 78 SW 626 Adenocarcinoma, ovary 79 Capan-1
Adenocarcinoma, pancreas, metastasis to liver 80 Capan-2
Adenocarcinoma, pancreas 81 DU 145 Carcinoma, prostate, metastasis
to brain 82 A-204 Rhabdomyosarcoma 85 Saos-2 Osteogenic sarcoma,
primary 86 SK-ES-1 Anaplastic osteosarcoma versus Ewing sarcoma,
bone 88 SK-LMS-1 Leiomyosarcoma, vulva, primary 91 SW 684
Fibrosarcoma 92 SW 872 Liposarcoma 93 Sw 982 Axilla synovial
sarcoma 94 Sw 1353 Chondrosarcoma, humerus 96 U-2 OS Osteogenic
sarcoma, bone primary 102 Malme-3 Skin fibroblast 103 KATO III
Gastric carcinoma 104 Cate-1B Embryonal carcinoma, testis,
metastasis to lymph node 105 Tera-1 Embryonal carcinoma, malignancy
consistent with metastasis to lung 106 Tera-2 Ernbryonal carcinoma,
malignancy consistent with, metastasis to lung 107 SW579 Thyroid
carcinoma 111 AN3 CA Endometrial adenocarcinoma, metastatic 112
HEC-1-A Endometrial adenocarcinoma 113 HEC-1-B Endometrial
adenocarcinoma 114 SK-UT-1 Uterine, mixed mesodermal tumor,
consistent with leiomyosarcoma grade III 115 SK-UT-1B Uterine,
mixed mesodermal tumor, consistent with lelomyosarcoma grade III
117 Sw 954 Squamous cell carcinoma, vulva 118 SW 962 Carcinoma,
vulva, lymph node metastasis 119 NCI-H69 Small cell carcinoma, lung
120 NCI-H128 Small cell carcinoma, lung 121 BT-483 Ductal
carcinoma, breast 122 BT-549 Ductal carcinoma, breast 123 DU4475
Metastatic cutaneous nodule, breast carcinoma 124 HBL-100 Breast
125 Hs 578Bst Breast, normal 126 Hs 578T Ductal carcinoma, breast
127 MDA-MB-330 Carcinoma, breast 128 MDA-MB-415 Adenocarcinoma,
breast 129 MDA-MB-435S Ductal carcinoma, breast 130 MDA-MB-436
Adenocarcinoma, breast 131 MDA-MB-453 Carcinoma, breast 132
MDA-MB-468 Adenocarcinoma, breast 133 T-47D Ductal carcinoma,
breast, pleural effusion 134 Hs 766T Carcinoma, pancreas,
metastatic to lymph node 135 Hs 746T Carcinoma, stomach, metastatic
to left leg 137 Hs 695T Amelanotic melanoma, metastatic to lymph
node 138 Hs 683 Glioma 140 Hs 294T Melanoma, metastatic to lymph
node 142 Hs 602 Lymphoma, cervical 144 JAR Choriocarcinoma,
placenta 146 Hs 445 Lymphoid, Hodgkin's disease 147 Ha 700T
Adenocarcinoma, metastatic to pelvis 148 H4 Neuroglioma, brain 151
Hs 696 Adenocarcinoma primary, unknown, metastatic to bone-sacrum
152 Hs 913T Fibrosarcoma, metastatic to lung 153 Hs 729
Rhabdomyosarcoma, left leg 157 FHs 738Lu Lung, normal fetus 158 FHs
173We Whole embryo, normal 160 FHs 738B1 Bladder, normal fetus 161
NIH:0VCAR-3 Ovary, adenocarcinoma 163 Hs 67 Thymus, normal 166
RD-ES Ewing's sarcoma 168 ChaGo K-1 Bronchogenic carcinoma,
subcutaneous metastasis, human 169 WERI-Rb-1 Retinoblastoma 171
NCI-H446 Small cell carcinoma, lung 172 NCI-H209 Small cell
carcinoma, lung 173 NCI-H146 Small cell carcinoma, lung 174
NCI-H441 Papillary adenocarcinoma, lung 175 NCI-H82 Small cell
carcinoma, lung 176 H9 T-cell lymphoma 177 NCI-H460 Large cell
carcinoma, lung. 178 NCI-H596 Adenosquamous carcinoma, lung 179
NCI-H676B Adenocarcinoma, lung 180 NCI-H345 Small cell carcinoma,
lung 181 NCI-H820 Papillary adenocarcinoma, lung 182 NCI-H520
Squamous cell carcinoma, lung 183 NCI-H661 Large cell carcinoma,
lung 184 NCI-H510A Small cell carcinoma, extra- pulmonary origin,
metastatic 185 D283 Med Medulloblastoma 186 Daoy Medulloblastoma
187 D341 Med Medulloblastoma 188 AML-193 Acute monocyte leukemia
189 MV4-11 Leukemia biphenotype
[0054] Tissue Factor
[0055] According to the invention, any Tissue Factor polypeptide
that can initiate thrombosis and that includes the extracellular
domain of Tissue factor can be used as the Tissue Factor domain of
the present Selective Tissue Vascular Thrombogens. The Tissue
Factor polypeptide can be mutant or wild type. The Tissue Factor
polypeptide can include all of the extracellular domain or part of
it. Preferably, the Tissue Factor polypeptide is not the
full-length native Tissue Factor. For example, the Tissue Factor
polypeptide generally lacks the cytoplasmic domain, and may have
none or only a part of the transmembrane domain.
[0056] Tissue Factor is the major receptor for initiating
thrombogenic (blood coagulation) cascades (Davie, et al. 1991).
Human Tissue Factor has been cloned and is available to those of
skill in the art (Morrissey et al., 1987; Edgington et al., 1991;
U.S. Pat. No. 5,110,730). In certain early studies, the same
protein currently identified as human Tissue Factor may be referred
to as human Tissue Factor heavy chain protein or the heavy chain of
Tissue Factor. The gene encodes a polypeptide precursor of 295
amino acids in length, which includes a peptide leader with
alternative cleavage sites, which is lead to the formation of a
protein of 263 amino acids in length. Mature Tissue Factor is a
single chain, 263 amino acid membrane glycoprotein (SEQ ID NO: 2),
and its primary sequence has structural similarity with the
cytokine receptor family (Edgington et al., 1991). The recombinant
expression of human Tissue Factor in CHO cells has been reported to
lead to the production of Tissue Factor at a level that is
described as being one of the highest expression levels reported
for a recombinant transmembrane receptor following production in
mammalian cells (Rehemtulla et al., 1991).
[0057] The amino acid sequence of the precursor form of human
Tissue Factor (SEQ ID NO: 1) is provided below:
2 -32 METPAWPRVP RPETAVARTL LLGWVFAQVA GA 1 SGTTNTVAAY NLTWKSTNFK
TILEWEPKPV NQVYTVQIST 41 KSGDWKSKCF YTTDTECDLT DEIVKDVKQT
YLARVFSYPA 81 GNVESTGSAG EPLYENSPEF TPYLETNLGQ PTIQSFEQVG 121
TKVNVTVEDE RTLVRRNNTF LSLRDVFGKD LIYTLYYWKS 161 SSSGKKTAKT
NTNEFLIDVD KGENYCFSVQ AVIPSRTVNR 201 KSTDSPVECM GQEKGEFREI
FYIIGAVVFV VIILVIILAI 241 SLHKCRKAGV GQSWKENSPL NVS
[0058] The amino acid sequence of the mature form of human Tissue
Factor (SEQ ID NO: 2) is provided below:
3 1 SGTTNTVAAY NLTWKSTNFK TILEWEPKPV NQVYTVQIST 41 KSGDWKSKCF
YTTDTECDLT DEIVKDVKQT YLARVFSYPA 81 GNVESTGSAG EPLYENSPEF
TPYLETNLGQ PTIQSFEQVG 121 TKVNVTVEDE RTLVRRNNTF LSLRDVFGKD
LIYTLYYWKS 161 SSSGKKTAKT NTNEFLIDVD KGENYCFSVQ AVIPSRTVNR 201
KSTDSPVECM GQEKGEFREI FYIIGAVVFV VIILVIILAI 241 SLHKCRKAGV
GQSWKENSPL NVS
[0059] The amino acid sequence of the extracellular domain of human
Tissue Factor (SEQ ID NO: 3), which is sometimes called TF1-219, is
provided below:
4 1 SGTTNTVAAY NLTWKSTNFK TILEWEPKPV NQVYTVQIST 41 KSGDWKSKCF
YTTDTECDLT DEIVKDVKQT YLARVFSYPA 81 GNVESTGSAG EPLYENSPEF
TPYLETNLGQ PTIQSFEQVG 121 TKVNVTVEDE RTLVRRNNTF LSLRDVFGKD
LIYTLYYWKS 161 SSSGKKTAKT NTNEFLIDVD KGENYCFSVQ AVIPSRTVNR 201
KSTDSPVECM GQEKGEFRE
[0060] The amino acid sequence of a slightly shorter extracellular
domain of human Tissue Factor (SEQ ID NO: 4), which is sometimes
called TF1-218, is provided below:
5 1 SGTTNTVAAY NLTWKSTNFK TILEWEPKPV NQVYTVQIST 41 KSGDWKSKCF
YTTDTECDLT DEIVKDVKQT YLARVFSYPA 81 GNVESTGSAG EPLYENSPEF
TPYLETNLGQ PTIQSFEQVG 121 TKVNVTVEDE RTLVRRNNTF LSLRDVFGKD
LIYTLYYWKS 161 SSSGKKTAKT NTNEFLIDVD KGENYCFSVQ AVIPSRTVNR 201
KSTDSPVECM GQEKGEFR
[0061] A slightly truncated extracellular domain of human Tissue
Factor that is sometimes called TF3-219 (SEQ ID NO: 5), because it
does not have the first two amino acids of SEQ ID NO: 3, can also
be used as a Tissue Factor polypeptide in the Tissue-Selective
Vascular Thrombogens of the invention. This TF3-219 polypeptide has
SEQ ID NO: 5, provided below:
6 1 TTNTVAAYNL TWKSTNFKTI LEWEPKPVNQ VYTVQISTKS 41 GDWKSKCFYT
TDTECDLTDE IVKDVKQTYL ARVFSYPAGN 81 VESTGSAGEP LYENSPEFTP
YLETNLGQPT IQSFEQVGTK 121 VNVTVEDERT LVRRNNTFLS LRDVFGKDLI
YTLYYWKSSS 161 SGKKTAKTNT NEFLIDVDKG ENYCFSVQAV IPSRTVNRKS 201
TDSPVECMGQ EKGEFRE
[0062] A similar amino acid sequence of a slightly truncated
extracellular domain of human Tissue Factor that is sometimes
called TF3-218 (SEQ ID NO: 6), because it does not have the first
two amino acids of SEQ ID NO: 3, can also be used as a Tissue
Factor polypeptide in the Tissue-Selective Vascular Thrombogens of
the invention. This TF3-218 polypeptide has SEQ ID NO: 6, provided
below:
7 1 TTNTVAAYNL TWKSTNFKTI LEWEPKPVNQ VYTVQISTKS 41 GDWKSKCFYT
TDTECDLTDE IVKDVKQTYL ARVFSYPAGN 81 VESTGSAGEP LYENSPEFTP
YLETNLGQPT IQSFEQVGTK 121 VNVTVEDERT LVRRNNTFLS LRDVFGKDLI
YTLYYWKSSS 161 SGKKTAKTNT NEFLIDVDKG ENYCFSVQAV IPSRTVNRKS 201
TDSPVECMGQ EKGEFR
[0063] Moreover, the C-terminal end of the Tissue Factor
polypeptide can be manipulated as desired by one of skill in the
art. For example, the transmembrane domain of Tissue Factor starts
at about amino acid 220 and ends at about amino position 241. This
domain and the cytoplasmic sequences that lie C-terminal to this
domain can be removed and replaced with other polypeptide
sequences, for example, other tranmembrane domains. In addition, a
peptide comprising approximate amino acid position 211 to
approximate amino acid position 219 can be removed and replaced
with any convenient peptide sequence that can link a transmembrane
domain to the active portion of Tissue Factor. Any such
manipulations can be performed to modify or enhance the activity or
membrane association propertied of the STVT, so long as the Tissue
Factor domain retains activity and is capable of initiated a
thrombogenic response.
[0064] A number of domains can be used to facilitate association of
Tissue Factor with a selected cellular membrane. For example, a
membrane association domain can be provided by the Selective
Binding Domain or by any membrane protein or member of the
Superfamily of Hematopoietic-Cytokine Receptors selected by one of
skill in the art. In one embodiment, the Selective Tissue Vascular
Thrombogens of the invention includes the entire transmembrane
domain of human Tissue Factor, or a portion thereof. The amino acid
sequence of the transmembrane domain of human Tissue Factor (SEQ ID
NO: 7) is provided below:
8 FYIIGAVVFV VIILVIILAI SL
[0065] Tissue Factor is a transmembrane cell surface receptor.
Tissue Factor functions as the receptor and requisite cofactor for
Factors VII and VIIa. Tissue Factor binds Factor VIIa to form a
proteolytically active binary complex on the cell surface (Ruf and
Edgington, 1991 b, 1994; Ruf et al., 1991, 1992a, 1992b). This
complex rapidly activates the serine protease zymogens Factors IX
and X by limited proteolysis, leading to the formation of thrombin
and, then to the conversion of plasma fibrinogen to fibrin
resulting in gelation of plasma and blood.
[0066] FIG. 1A provides a structural model of the ternary complex
of Tissue Factor (having a transmembrane and a cytoplasmic domain)
with Factor VIIa (red molecule to the left) and Factor X (yellow
molecule to the right that extends down to become) as this complex
becomes associated with a cell surface. The transmembrane domain of
native Tissue Factor spans the cell membrane and ensures proper
positioning of both Factor VIIa and Factor X (or Factor IX) on the
cell surface. The interaction of the N-terminal Gla domain of both
Factor VIIa and Factor X with the cell membrane is critical for the
full thrombogenic activity of this complex.
[0067] A limited number of cells constitutively express Tissue
Factor. Lung and central nervous system tissues contain high levels
of Tissue Factor activity, with Tissue Factor being found in
bronchial mucosa and alveolar epithelial cells in the lung and in
glial cells and astrocytes in the nervous system. Expression of
Tissue Factor has also been reported in cardiac myocytes, renal
glomeruli, and in certain epithelial or mucosal tissues of the
intestine, bladder and respiratory tract. Over expression of Tissue
Factor has been linked to thrombotic diseases and sepsis (Drake,
Morrissey et al. 1989; Levi, ten Cate et al. 1994). Tissue Factor
expression on endothelial cells and monocytes is induced by
exposure to inflammatory cytokines and bacterial lipopolysaccaride
(Drake and Pang 1989; Oeth, Pany et al. 1994). There can also be
very small amounts of Tissue Factor associated with membrane
vesicles shed by cells into the blood. Tissue Factor is generally
constitutively expressed at tissue barriers between body tissues
and the external environment (Drake et al., 1989; Ruf and
Edgington, 1994). The expression of Tissue Factor in this manner
acts as an envelope that allows Tissue Factor to arrest
bleeding.
[0068] The disruption of the Tissue Factor gene in mice revealed an
unexpected role for Tissue Factor in supporting vascular
development (Carmeliet, Mackman et al. 1996). Beside the roles it
has in hemostasis and thrombosis, Tissue Factor is also implicated
in signal transduction (Zioncheck, Roy et al. 1992), cellular
adhesion (Ott, Fischer et al. 1998), and the tumor metastasis and
angiogenesis (Mueller and Ruf 1998).
[0069] Tissue Factor is typically not expressed to any significant
degree on blood cells or on the surface of endothelial cells that
form the vasculature, but such expression can be induced in
endothelial cells and monocytes within the vasculature by
infectious agents and certain inflammatory processes. Monocytes,
for example, are induced to express Tissue Factor by cytokines and
T cells. Expression of Tissue Factor in the vasculature can result
in disseminated intravascular coagulation or localized initiation
of blood clots or thrombogenesis. In this context, it is important
to note that Tissue Factor must be available at all sites of the
body where hemostasis would be necessary following tissue damage,
infection or other insults.
[0070] Tissue Factor is the major initiator of the blood
coagulation protease cascades and is generally not in direct
contact with the blood under physiologically normal conditions
(Osterud et al., 1986; Nemerson, 1988; Broze, 1992; Ruf and
Edgington, 1994). After vascular damage or activation by certain
cytokines or endotoxin, however, Tissue Factor is exposed to the
blood by the exposure of (sub)endothelial tissue and cells (Weiss
et al., 1989), by induction within the endothelium, or by certain
blood cells (Warr et al., 1990). Tissue Factor then complexes with
Factor VII and VIIa, which under normal conditions circulate at low
concentrations in the blood (Wildgoose et al., 1992). The Tissue
Factor:Factor VII complex is converted to a Tissue Factor:Factor
VIIa complex. The Tissue Factor/Factor VIIa complex starts the
coagulation cascade through the activation of Factor X to Factor
Xa. Ultimately, the cascade results in formation of thrombin that
produces fibrin.
[0071] For this sequence of events to occur, the Tissue
Factor:Factor VIIa complex must be associated with a supportive
phospholipid membrane surface in order for efficient assembly of
the coagulation-initiation complexes with Factors IX or X (Ruf and
Edgington, 1991a; Ruf et al., 1992c; Paborsky et al., 1991; Bach et
al., 1986; Krishnaswamy et al., 1992; ten Cate et al., 1993). The
association of Tissue Factor with an anionic phospholipid membrane
increases the coagulative activity of this complex by promoting the
proper orientation of Factor VIIa relative to Tissue Factor through
the interaction of Gla domain of Factor VIIa with phospholipid.
This enhances the binding of Factor VIIa to Tissue Factor,
facilitates the catalytic conversion of Factor VII to Factor VIIa,
and enhances the activity of Tissue Factor:Factor VIIa toward its
substrates, Factor X and Factor IX. It also provides a cellular
membrane binding for Factor X and Factor IX.
[0072] A recombinant form of Tissue Factor has been constructed
that contains only the cell surface or extracellular domain (Ruf et
al., 1991b; Stone, et al., 1995) and that lacks the transmembrane
and cytoplasmic regions of Tissue Factor. This truncated Tissue
Factor is 219 amino acids in length and is a soluble protein with
approximately 10.sup.5 times less factor X-activating activity than
native Tissue Factor in an appropriate phospholipid membrane
environment (Ruf, et al., 1991b). This difference in activity is
related to the association of Tissue Factor and the lack of
membrane-association by truncated Tissue Factor. The Tissue
Factor:VIIa complex binds and activates Factors IX and X far more
efficiently when associated with a negatively charged phospholipid
surface (Ruf, et al., 1991b; Paborsky, et al., 1991). Consequently,
the native transmembrane Tissue Factor is 100,000 fold more active
than the soluble Tissue Factor extracellular domain. In order to
achieve site-selective induction of thrombosis to occlude undesired
vessels under pathologic conditions using Tissue Factor, a soluble
Tissue Factor molecule that retains coagulative function upon
proper positioning onto a cell surface structure is desirable.
[0073] However, according to the present invention, the
extracellular domain of Tissue Factor, without the natural
transmembrane and cytoplasmic regions of Tissue Factor, can promote
blood coagulation when properly associated with a cellular membrane
by any "Selective Binding Domain."
[0074] Selective Binding Domains
[0075] A Selective Binding Domain is a peptide, peptidyl analogue
or polypeptide that can associate with a cellular membrane, through
direct interaction with the membrane or through interaction with a
protein present on the membrane, or both. Association with the
cellular membrane by the Selective Binding Domain need only be
transient, however, it must be selective so that the Selective
Tissue Vascular Thrombogen can provide targeted, localized
thrombosis.
[0076] One or more Selective Binding Domains are associated or
integrated with one or more Tissue Factor polypeptides to form a
Selective Tissue Vascular Thrombogen. Association between the
Tissue Factor polypeptide(s) and the Selective Binding Domain(s)
can be via a covalent bond, or via any other stable interaction,
such as by hydrogen bonding.
[0077] FIG. 1B shows a structural model of a ternary complex of a
Tissue-Selective Vascular Thrombogen of the invention where the
Tissue Factor polypeptide (in the middle) is associated with Factor
VIIa (on the left) and Factor X (on the right). The N-terminal
extracellular domain of Tissue Factor is fused with a Selective
Binding Domain (arrow in the upper left corner of FIG. 1B) to form
a novel Tissue-Selective Vascular Thrombogen. The extreme portion
of the N-terminus of Tissue Factor is not involved in its function.
Hence, addition of another molecule or domain to the N-terminus of
a Tissue Factor polypeptide is possible. In this invention, a
Selective Binding Domain can be added to properly associate and
physically align the Tissue Factor polypeptide with the cell
surface. Attachment of such a Selective Binding Domain to its
N-terminus does not adversely affect the conformation or the
function of the selected Tissue Factor polypeptide. Factor VII, or
its activated form, Factor VIIa, and Factor X, or Factor IX, can
therefore interact with the Tissue Factor domain when aligned on a
functionally supportive region of an anionic cell surface. Such
interaction permits formation of a complex that has a conformation
very similar to the structure of a native Tissue Factor:Factor
VIIa:Factor Xa complex. The thrombogenic activity of this complex
is substantially unaffected by the incorporated Selective Binding
Domain.
[0078] A Selective Binding domain can be a ligand for a cellular
receptor, a membrane-associated domain for any cell membrane
protein known to one of skill in the art, an inhibitor for a
membrane-associated protein, a component of tumor vasculature, a
component that binds to, or is generally associated with, tumor
cells, a component that binds to, or is generally associated with,
tumor vasculature, a component of the tumor extracellular matrix or
stroma, a cell found within the tumor vasculature or any peptide or
polypeptide that preferentially interacts with a cellular membrane.
In general the Selective Binding domain is preferably not an
antibody.
[0079] Selective Binding domains can be made to bind to any
relatively specific marker on the tumor cell, for example,
endoglin, integrin, VEGF receptor, PSMA and the like. Many
so-called "tumor antigens" have been described, any one which could
be employed as a target to which the Selective Binding domain may
bind. A large number of exemplary solid tumor-associated antigens
are listed herein in Table 2.
9TABLE 2 Marker Antigens of Solid Tumors Antigen Identity/
Monoclonal Tumor Site Characteristics Antibodies Reference
GYNECOLOGICAL GY CA 125 >200 kiD OC 125 Kabawat et al., mucin GP
1983; Szymendera, 1986 Ovarian 80 Kd GP OC 133 Masuko et al, Cancer
Res., 1984 Ovarian SGA 360 Kd GP OMI de Krester et al., 1986
Ovarian High M.sub.r mucin MO v1 Miotti et al, Cancer Res., 1985
Ovarian High M.sub.r mucin/ MO v2 Miotti Ct at, glycolipid Cancer
Res., 1985 Ovarian NS 3C2 Tsuji et at., Cancer Res., 1985 Ovarian
NS 4C7 Tsuji et at., Cancer Res., 1985 Ovarian High M.sub.r mucin
ID.sub.3 Gangopadhyay et al., 1985 Ovarian High M.sub.r mucin
DU-PAN-2 Lan et al., 1985 GY 7700 Kd GP F 36/22 Croghan Ct at.,
1984 Ovarian `gp 68` 48 Kd 4F.sub.7/A.sub.10 Bhattacharya et al.,
1984 GY 40, 42kD GP OV-TL3 Poels et al., 1986 GY `TAG-72` High
B72.3 Thor et al., 1986 M.sub.r mucin Ovarian 300-400 Kd GP
DF.sub.3 Kufe et al., 1984 Ovarian 60 Kd GP 2C.sub.8/2F.sub.7
Bhattacharya et al., 1985 GY 105 Kd GP MF 116 Mattes et al., 1984
Ovarian 38-40 kD GP MOv18 Miotti et al., 1987 GY CEA 180 Kd GP CEA
11-H5 Wagener et al., 1984 Ovarian CA 19-9 or GICA CA 19-9 Atkinson
et al., (1116NS 1982 19-9) Ovarian `PLAP` 67 Kd H17-E2 McDicken et
al., GP 1985 Ovarian 72 Kd 791T/36 Perkins et al., 1985 Ovarian 69
Kd PLAP NDOG.sub.2 Sunderland et al., 1984 Ovarian unknown M.sub.r
H317 Johnson et al., PLAP 1981 Ovarian p185.sup.HER2 4D5, 3H4,
Shepard et al., 7C2, 6E9, 1991 2C4, 7F3, 2H11, 3E8, 5B8, 7D3, SB8
uterus ovary HMFG-2 HMFG2 Epenetos et al., 1982 GY HMFG-2 3.14.A3
Butchell et al., 1983 BREAST 330-450 Kd GP DF3 Hayes Ct al., 1985
NS NCRC-11 Ellis et al., 1984 37kD 3C6F9 Mandeville et al., 1987 NS
MBE6 Teramoto et al., 1982 47 Kd GP MAC 40/43 Kjeldsen et al., 1986
High M.sub.r GP EMA Sloane et al., 1981 High M.sub.r GP HMFG1
Arklie et al., HFMG2 1981 NS 3.15.C3 Arklie et al., 1981 NS M3, M8,
Foster et al., M24 1982 1 (Ma) blood M18 Foster et al., group Ags
1984 NS 67-D-11 Rasmussen et al., 1982 oestrogen D547Sp, Kinsel et
al., receptor D75P3, 1989 H222 EGF Receptor Anti-EGF Sainsbury et
al., 1985 Laminin Receptor LR-3 Horan Hand et al., 1985 Erb B-2
p185 TA1 Gusterson et al., 1988 NS H59 Hendler et al., 1981 126 Kd
GP 10-3D-2 Soule et al., 1983 NS HmAB1,2 Imam et al., 1984; Schlom
et al., 1985 NS MBR 1,2,3 Menard et al., 1983 95 Kd 24.17.1
Thompson et al., 1983 100 Kd 24.17.2 Croghan et al., (3E1.2) 1983
NS F36/22.M7/ Croghanet al., 105 1984 24 Kd C11,G3, Adams et al.,
H7 1983 90 Kd GP B6.2 Colcher Ct al., 1981 CEA & 180 Kd B1.1.
Colcher et al., GP 1983 Colonic & Cam 17.1 Imperial Cancer
pancreatic mucin Research similar to Ca 19-9 Technology MAb listing
milk mucin core SM3 Imperial Cancer protein Research Technology MAb
listing milk mucin core SM4 Imperial Cancer protein Research
Technology MAb listing Affinity-purified C-Mul (566) Imperial
Cancer milk mucin Research Technology MAb listing p185.sup.HER2
4D5, 3H4, Shepard et al., 7C2, 6E9, 1991 2C4, 7F3, 2H11, 3E8, 5B8,
7D3, SB8 CA 125 > 200 Kd OC 125 Kabawat et al., GP 1985 High
M.sub.r mucin/ MO v2 Miotti et al., glycoprotein 1985 High M.sub.r
mucin DU-PAN-2 Lan et al., 1984 `gp48` 48 Kd GP 4F.sub.7/7A.sub.10
Bhattacharya et al., 1984 300-400 Kd GP DF.sub.3 Kufe et al., 1984
`TAG-72` high B72.3 Thor et al., 1986 M.sub.r mucin `CEA` 180 Kd
cccccCEA Wagener et al., GP 11 1984 `PLAP` 67 Kd H17-E2 McDicken et
al., GP 1985 HMFG-2 > 400 3.14.A3 Burchell et al., Kd GP 1983 NS
FO23C5 Riva et al., 1988 COLORECTAL TAG-72 High M.sub.r B72.3
Colcher et al., mucin 1987 GP37 (17-1A) Paul et al., 1986
1083-17-1A Surface GP CO17-1A LoBuglio et al., 1988 CEA ZCE-025
Patt et al., 1988 CEA AB2 Griffin et al., 1988a Cell surface AG
HT-29-15 Cohn et al., 1987 secretory 250-30.6 Leydem et al.,
epithelium 1986 Surface 44 .times. 14 Gallagher et al.,
glycoprotein 1986 NS A7 Takahashi et al., 1988 NS GA73.3 Munz et
al., 1986 NS 791T/36 Farrans et al., 1982 cell membrane & 28A32
Smith et al., cytoplasmic Ag 1987 CEA & vindesine 28.19.8
Corvalen, 1987 gp72 x MMCO- Byers et al., 791 1987 High M.sub.r
mucin DU-PAN-2 Lan et al., 1985 High M.sub.r mucin ID.sub.3
Gangopadhyay et al., 1985 CEA 180 Kd GP CEA 11-H5 Wagener et al.,
1984 60 Kd GP 2C.sub.8/2F.sub.7 Bhattacharya et al., 1985 CA-19-9
(or C-19-9 Atkinson et al., GICA) (1116NS 1982 19-9) Lewis a PR5C5
Imperial Cancer Research Technology Mab Listing Lewis a PR4D2
Imperial Cancer Research Technology Mab Listing Colonic mucus PR4D1
Imperial Cancer Research Technology Mab Listing MELANOMA p97.sup.a
4.1 Woodbury et al., 1980 p97.sup.a 8.2 M.sub.17 Brown, et al.,
1981a p97.sup.b 96.5 Brown, et al., 1981a p97.sup.c 118.1, Brown,
et al., 133.2, 1981a (113.2) p97.sup.c L.sub.1,L.sub.10,R.sub.10
Brown, et al., (R.sub.19) 1981b p97.sup.d I.sub.12 Brown, et al.,
1981b p97.sup.e K.sub.5 Brown et al., 1981b p155 6.1 Loop et al.,
1981 G.sub.D3 disialogan- R24 Dippold et al., glioside 1980 p210,
p60, p250 5.1 Loop et. al., 1981 p280 p440 225.28S Wilson et al.,
1981 GP 94, 75, 70 & 465.12S Wilson et al., 25 1981 P240-P250,
P450 9.2.27 Reisfeld et al., 1982 100, 77, 75 Kd F11 Chee et al.,
1982 94 Kd 376.96S Imai et al., 1982 4 GP chains 465.12S Imai et
al., 1982; Wilson et al., 1981 GP 74 15.75 Johnson &
Reithmuller, 1982 GP49 15.95 Johnson & Reithmuller, 1982 230 Kd
Mel-14 Carrel et al., 1982 92 Kd Mel-12 Carrel et al., 1982 70 Kd
Me3-TB7 Carrel et al. 1982 HMW MAA 225.28SD Kantor et al., similar
to 9.2.27 1982 AG HMW MAA 763.24TS Kantor et al., similar to 9.2.27
1982 AG GP95 similar to 705F6 Stuhlmiller et 376.96S 465.125 al.,
1982 GP125 436910 Saxton et al., 1982 CD41 M148 Imperial Cancer
Research Technology Mab listing GASTROINTESTINAL High M.sub.r mucin
ID3 Gangopadhyay et al., 1985 gall bladder, pancreas, High M.sub.r
mucin DU-PAN-2 Lan et al., 1985 stomach pancreas NS OV-TL3 Poels et
al., 1984 pancreas, stomach, `TAG-72` high B72.3 Thor et al., 1986
oesophagus M.sub.r mucin stomach `CEA` 180 Kd CEA 11-H5 Wagener et
al., GP 1984 pancreas HMFG-2 > 400 3.14.A3 Burchell et al., Kd
GP 1983 G.I. NS C COLI Lemkin et al., 1984 pancreas, stomach CA
19-9 (or CA-19-9 Szymendera, GICA) (1116NS 1986 19-9) and CA50
pancreas CA125 GP OC125 Szymendera, 1986 LUNG non-small cell lung
p185.sup.HER2 4D5, 3H4, Shepard et al., carcinoma 7C2, 6E9, 1991
2C4, 7F3, 2H11, 3E8, 5B8, 7D3, SB8 high M.sub.r mucin/ MO v2 Miotti
et al., glycolipid 1985 `TAG-72` high B72.3 Thor et al., 1986
M.sub.r mucin high M.sub.r mucin DU-PAN-2 Lan et al., 1985 `CEA`
180 kD GP CEA 11-H5 Wagener et al., 1984 Malignant Gliomas
cytoplasmic MUG 8-22 Stavrou, 1990 antigen from 85HG-22 cells cell
surface Ag MUG 2-63 Stavrou, 1990 from 85HG-63 cells cell surface
Ag MUG 2-39 Stavrou, 1990 from 85HG-63 cells cell surface Ag MUG
7-39 Stavrou, 1990 from 85HG-63 cells MISCELLANEOUS p53 PAb 240,
Imperial Gancer PAb 246, Research PAb 1801 Technology MaB Listing
small round cell neural cell ERIC. 1 Imperial Gancer tumors
adhesion Research molecule Technology MaB Listing medulloblastoma
M148 Imperial Gancer neuroblastoma Research rhabdomyosarcoma
Technology MaB Listing neuroblastoma FMH25 Imperial Cancer Research
Technology MaB Listing renal cancer & p155 6.1 Loop et al.,
1981 glioblastomas Bladder & laryngeal "Ca Antigen"350- CA1
Ashall et al., cancers 390 kD 1982 neuroblastoma GD2 3F8 Cheung et
al., 1986 Prostate gp48 48 kD GP 4F.sub.7/7A.sub.10 Bhattacharya et
al., 1984 Prostate 60 kD GP 2C.sub.8/2F.sub.7 Bhattacharya et al.,
1985 Thyroid `CEA` 180 kD CEA 11-H5 Wagener et al., GP 1984
[0080] abbreviations: Abs, antibodies; Ags, antigens; EGF,
epidermal growth factor; GI, gastrointestinal; GICA,
gastrointestinal-associated antigen; GP, glycoprotein; GY,
gynecological; HMFG, human milk fat globule; Kd, kilodaltons; Mabs,
monoclonal antibodies; M.sub.r, molecular weight; NS, not
specified; PLAP, placental alkaline phosphatase; TAG,
tumor-associated glycoprotein; CEA, carcinoembryonic antigen. Note:
the CA 199 Ag (GICA) is sialosylfucosyllactotetraosylceramide, also
termed sialylated Lewis pentaglycosyl ceramide or sialyated lacto
N-fucopentaose II; p97 Ags are believed to be chondroitin sulphate
proteoglycan; antigens reactive with Mab 9.2.27 are believed to be
sialylated glycoproteins associated with chondroitin sulphate
proteoglycan; unless specified, GY can include cancers of the
cervix, endocervix, endometrium, fallopian tube, ovary, vagina or
mixed Mullerian tumor; unless specified GI can include cancers of
the liver, small intestine, spleen, pancreas, stomach and
oesophagus.
[0081] In one embodiment, the Selective Binding Domain is an
inhibitor of prostate specific membrane antigen (PSMA) or folate
glutamate hydrolase. Prostate specific membrane antigen (PSMA) is a
signal marker for prostate that is over-expressed in prostate
carcinoma, especially in advanced tumors. The PSMA protein is a
glutamyl preferring carboxypeptidase that can release glutamate
with either gamma or alpha linkages. New data indicates that PSMA
is selectively expressed and apparently present on the endothelial
surface of tumor microvasculature.
[0082] According to the invention, endothelial-like tumor cells
that express PSMA can undergo a novel differentiation process
termed "vasculogenic mimicry." Such vasculogenic mimicry occurs
when such endothelial-like tumor cells form vessels within solid
prostate tumors. These tumor vessels connect with the normal
circulatory system and may provide blood nutrients and oxygen to
the interior of solid tumors. Therefore, according to the
invention, proteins that are expressed on endothelial-like solid
tumor cells can serve as recognition sites or targets for the
Selective Tissue Vascular Thrombogens of the invention.
[0083] PSMA is one such target for the Selective Tissue Vascular
Thrombogens of the invention. According to the invention, any
molecule that can bind to PSMA can be used as a Selective Binding
Domain for a Selective Tissue Vascular Thrombogen that can be used
to treat prostate tumors. Selective Binding Domains that can be
used to target PSMA include PSMA inhibitors and modified
substrates, for example, the dipeptide Asp-.beta., linked-Glu
(D.beta.E), and N-succinyl-glutamic acid. The Asp-.beta. linked-Glu
dipeptide is a suicidal inhibitor of the PSMA protease. According
to the invention, a Asp-.beta. linked-Glu-biotin:avidin-Tissue
Factor:VIIa thrombogen complex induces tumor infarction in PSMA
expressing prostate tumors without harming the animal.
[0084] In another embodiment, inhibitors of N-Acetylated
.alpha.-Linked Acidic Dipeptidase (NAALADase) are used as the
Selective Binding Domain to deliver the thrombogen to the selected
target. Examples of such NAALADase inhibitors include phosphonate
moieties, such as 2-(phosphonomethyl)pentanedioic acid. Further
examples include the following:
[0085] 2-Methylhydroxyphosphinyl oxypentanedioic acid;
[0086] 2-Ethylhydroxyphosphinyl oxypentanedioic acid;
[0087] 2-Propylhydroxyphosphinyl oxypentanedioic acid;
[0088] 2-Butylhydroxyphosphinyl oxypentanedioic acid;
[0089] 2-Phenylhydroxyphosphinyl oxypentanedioic acid;
[0090] 2-(Phenylmethyl)hydroxyphosphinyl oxypentanedioic acid;
[0091] (2-Phenylethyl)methyl)hydroxyphosphinyl oxypentanedioic
acid.
[0092] Another group of NAALADase enzyme inhibitors that can be
used to deliver the present thrombogen contain phosphoramidates and
related groups, for example:
[0093] Methylhydroxyphosphinyl glutamic acid;
[0094] Ethylhydroxyphosphinyl glutamic acid;
[0095] Propylhydroxyphosphinyl glutamic acid;
[0096] Butylhydroxyphosphinyl glutamic acid;
[0097] Phenylhydroxyphosphinyl glutamic acid;
[0098] (Phenylmethyl)hydroxyphosphinyl glutamic acid;
[0099] ((2-Phenylethyl)methyl)hydroxyphosphinyl glutamic acid;
and
[0100] Methyl-N->Phenylhydroxyphosphinyl glutamic acid.
[0101] Another group of NAALADase enzyme inhibitors that can be
used to deliver thrombogens have a phosphinic acid group. Such
inhibitors contain any one of the following moieties:
[0102] 2-methylhydroxyphosphinyl methylpentanedioic acid;
[0103] 2-ethylhydroxyphosphinyl methylpentanedioic acid;
[0104] 2-propylhydroxyphosphinyl methylpentanedioic acid;
[0105] 2-butylhydroxyphosphinyl methylpentanedioic acid;
[0106] 2-cylobexylhydroxyphosphinyl methylpentanedioic acid;
[0107] 2-phenylhydroxyphosphinyl methylpentanedioic acid;
[0108] 2-(phenylmethyl) hydroxyphosphinyl methylpentanedioic
acid;
[0109] 2-((2-phenylethyl)methyl)hydroxyphosphinyl
methylpentanedioic acid;
[0110] 2-((3-phenylpropyl)methyl)hydroxyphosphinyl
methylpentanedioic acid;
[0111] 2-((3-phenylbutyl)methyl)hydroxyphosphinyl
methylpentanedioic acid;
[0112] 2-((2-phenylbutyl)methyl)hydroxyphosphinyl
methylpentanedioic acid;
[0113] 2-(4-phenylbutyl) hydroxyphosphinyl methylpentanedioic
acid;
[0114] 2-(aminomethyl) hydroxyphosphinyl methylpentanedioic
acid.
[0115] Certain sulfoxide and sulfone derivatives also act as
inhibitors of NAALADase enzymes and can be used to deliver the
thrombogen of the invention. Such inhibitors contain any one of the
following moieties:
[0116] 2-(sulfinyl)methylpentanedioic acid;
[0117] 2-(methylsulfinyl)methylpentanedioic acid;
[0118] 2-(ethylsulfinyl)methylpentanedioic acid;
[0119] 2-(propylsulfinyl)methylpentanedioic acid;
[0120] 2-(butylsulfinyl)methylpentanedioic acid;
[0121] 2-(phenylsulfinyl methylpentanedioic acid;
[0122] 2-(2-phenylethyl)sulfinyl methylpentanedioic acid;
[0123] 2-(3-phenylpropyl)sulfinyl methylpentanedioic acid;
[0124] 2-(4-pyridyl)sulfinyl methylpentanedioic acid; and
[0125] 2-(benzylsulfinyl)methylpentanedioic acid.
[0126] 2-(sulfonyl)methylpentanedioic acid;
[0127] 2-(methylsulfonyl)methylpentanedioic acid;
[0128] 2-(ethylsulfonyl)methylpentanedioic acid;
[0129] 2-(propylsulfonyl)methylpentanedioic acid;
[0130] 2-(butylsulfonyl)methylpentanedioic acid;
[0131] 2-(phenylsulfonyl)methylpentanedioic acid;
[0132] 2-(2-phenylethyl)sulfonyl methylpentanedioic acid;
[0133] 2-(3-phenylpropyl)sulfonyl methylpentanedioic acid;
[0134] 2-(4-pyridyl)sulfonylmethylpentanedioic acid; and
[0135] 2-(N-hydroxy)carbamoyl methylpentanedioic acid;
[0136] Yet another group of NAALADase inhibitors contain hydroxamic
acid moieties. Examples of such moieties include the following.
[0137] 2-(N-hydroxy-N-methyl)carbamoyl methylpentanedioic acid;
[0138] 2-(N-butyl-N-hydroxy)carbamoyl methylpentanedioic acid;
[0139] 2-(N-benzyl-N-hydroxy)carbamoyl methylpentanedioic acid;
[0140] 2-(N-hydroxy-N-phenyl)carbamoyl methylpentanedioic acid;
[0141] 2-(N-hydroxy-N-2-phenylethyl) carbamoylmethylpentanedioic
acid;
[0142] 2-(N-ethyl-N-hydroxy)carbamoyl methylpentanedioic acid;
[0143] 2-(N-hydroxy-N-propyl)carbamoylmethylpentanedioic acid;
[0144] 2-(N-hydroxy-N-3-phenylpropyl)carbamoyl methylpentanedioic
acid; and
[0145] 2-(N-hydroxy-N-4-pyridyl)carbamoyl methylpentanedioic
acid
[0146] 2-(benzylsulfonyl)methylpentanedioic acid.
[0147] One of skill in the art can readily prepare and integrate
these types of moieties and inhibitor molecules into the Selective
Tissue Vascular Thrombogens of the invention with one or more
Tissue Factor polypeptides using available procedures. See, e.g.,
U.S. Pat. Nos. 5,795,877; 5,863,536; 5,902,817; 5,968,915; and
5,880,112.
[0148] Another Selective Binding domain contemplated by the
invention is a peptide or polypeptide containing an Arg-Gly-Asp
(RGD) tripeptide sequence. Any RGD-containing Selective Binding
Domain that can bind to an integrin is contemplated by the
invention. A number of integrins exist and, according to the
invention, such integrin subtypes will all support the coagulative
activity of a Tissue Factor:RGD Selective Binding Domain construct.
For example, VLA is the major integrin expressed on the CHO cell
surface, and the Alpha V beta III integrins are reported to be
expressed on angiogenic tumor vessels. Inhibition of alpha V beta
III integrin with LM609 antibody was shown to inhibit angiogenesis
and tumor growth (Brooks, Montgomery et al. 1994). According to the
invention, RGD Selective Binding Domain:Tissue Factor constructs
can be used to occlude blood vessels within integrin-expressing
tumors.
[0149] One type of RGD bearing Selective Binding Domain useful in
the invention can be derived from the extra-cellular matrix
glycoprotein fibronectin. Fibronectin contains a number of domains
that mediate its association with integrin molecules. One region of
fibronectin that mediates its interaction with integrin is the
Central Cell Binding Domain (CCBD). This region contains a number
of homologous repeating polypeptide modules termed Fibronectin Type
III Repeats, each being about 90 amino acids residues long. An RGD
sequence, located in the tenth Fibronectin type III repeat, is a
key recognition site for several different integrins, including the
Alpha5beta 1 integrin family.
[0150] According to the invention, synthetic peptides having the
RGD sequence and the Fibronectin type III repeat domains 8-11 are a
useful Selective Binding Domains that can successfully confer
coagulative properties to the surface of the cells that are
otherwise inactive. Alternatively, one may use only the fibronectin
10.sup.th type III repeat domain.
[0151] In one embodiment, the Selective Binding Domain is selected
from human fibronectin, having, for example, SEQ ID NO: 8.
10 1 MVQPQSPVAV SQSKPGCYDN GKHYQINQQW ERTYLGNALV 41 CTCYGGSRGF
NCESKPEAEE TCFDKYTGNT YRVGDTYERP 81 KDSMIWDCTC IGAGRGRISC
TIANRCHEGG QSYKIGDTWR 121 RPHETGGYML ECVCLGNGKG EWTCKPIAEK
CFDHAAGTSY 161 VVGETWEKPY QGWMMVDCTC LGEGSGRITC TSRNRCNDQD 201
TRTSYRIGDT WSKKDNRGNL LQCICTGNGR GEWKCERHTS 241 VQTTSSGSGP
FTDVRAAVYQ PQPHPQPPPY GHCVTDSGVV 281 YSVGMQWLKT QGNKQMLCTC
LGNGVSCQET AVTQTYGGNS 321 NGEPCVLPFT YNGRTFYSCT TEGRQDGHLW
CSTTSNYEQD 361 QKYSFCTDHT VLVQTRGGNS NGALCHFPFL YNNHNYTDCT 401
SEGRRDNMKW CGTTQNYDAD QKFGFCPMAA HEEICTTNEG 441 VMYRIGDQWD
KQHDMGHMMR CTCVGNGRGE WTCIAYSQLR 481 DQCIVDDITY NVNDTFHKRH
EEGHMLNCTC FGQGRGRWKC 521 DPVDQCQDSE TGTFYQIGDS WEKYVHGVRY
QCYCYGRGIG 561 EWHCQPLQTY PSSSGPVEVF ITETPSQPNS HPIQWNAPQP 601
SHISKYILRW RPKNSVGRWK EATIPGHLNS YTIKGLKPGV 641 VYEGQLISIQ
QYGHQEVTRF DFTTTSTSTP VTSNTVTGET 681 TPFSPLVATS ESVTEITASS
FVVSWVSASD TVSGFRVEYE 721 LSEEGDEPQY LDLPSTATSV NIPDLLPGRK
YIVNVYQISE 761 DGEQSLILST SQTTAPDAPP DTTVDQVDDT SIVVRWSRPQ 801
APITGYRIVY SPSVEGSSTE LNLPETANSV TLSDLQPGVQ 841 YNITIYAVEE
NQESTPVVIQ QETTGTPRSD TVPSPRDLQF 881 VEVTDVKVTI MWTPPESAVT
GYRVDVIPVN LPGEHGQRLP 921 ISRNTFAEVT GLSPGVTYYF KVFAVSHGRE
SKPLTAQQTT 961 KLDAPTNLQF VNETDSTVLV RWTPPRAQIT GYRLTVGLTR 1001
RGQPRQYNVG PSVSKYPLRN LQPASEYTVS LVAIKGNQES 1041 PKATGVFTTL
QPGSSIPPYN TEVTETTIVI TWTPAPRIGF 1081 KLGVRPSQGG EAPREVTSDS
GSIVVSGLTP GVEYVYTIQV 1121 LRDGQERDAP IVNKVVTPLS PPTNLHLEAN
PDTGVLTVSW 1161 ERSTTPDITG YRITTTPTNG QQGNSLEEVV HADQSSCTFD 1201
NLSPGLEYNV SVYTVKDDKE SVPISDTIIP AVPPPTDLRF 1241 TNIGPDTMRV
TWAPPPSIDL TNFLVRYSPV KNEEDVAELS 1281 ISPSDNAVVL TNLLPGTEYV
VSVSSVYEQH ESTPLRGRQK 1321 TGLDSPTGID FSDTTANSFT VHWIAPRATI
TGYRIRHHPE 1361 HFSGRPREDR VPHSRNSITL TNLTPGTEYV VSIVALNGRE 1401
ESPLLIGQQS TVSDVPRDLE VVAATPTSLL ISWDAPAVTV 1441 RYYRITYGET
GGNSPVQEFT VPGSKSTATI SGLKPGVDYT 1481 ITVYAVTGRG DSPASSKPIS
INYRTEIDKP SQMQVTDVQD 1521 NSISVKWLPS SSPVTGYRVT TTPKNGPGPT
KTKTAGPDQT 1561 EMTIEGLQPT VEYVVSVYAQ NPSGESQPLV QTAVTNIDRP 1601
KGLAFTDVDV DSIKIAWESP QGQVSRYRVT YSSPEDGIHE 1641 LFPAPDGEED
TAELQGLRPG SEYTVSVVAL HDDMESQPLI 1681 GTQSTAIPAP TDLKFTQVTP
TSLSAQWTPP NVQLTGYRVR 1721 VTPKEKTGPM KEINLAPDSS SVVVSGLMVA
TKYEVSVYAL 1761 KDTLTSRPAQ GVVTTLENVS PPRRARVTDA TETTITISWR 1801
TKTETITGFQ VDAVPANGQT PIQRTIKPDV RSYTITGLQP 1841 GTDYKIYLYT
LNDNARSSPV VIDASTAIDA PSNLRFLATT 1881 PNSLLVSWQP PRARITGYII
KYEKPGSPPR EVVPRPRPGV 1921 TEATITGLEP GTEYTTYVIA LKNNQKSEPL
IGRKKTDELP 1961 QLVTLPHPNL HGPEILDVPS TVQKTPFVTH PGYDTGNGIQ 2001
LPGTSGQQPS VGQQNIFEEH GFRRTTPPTT ATPIRHRPRP 2041 YPPNVGQEAL
SQTTISWAPF QDTSEYIISC HPVGTDEEPL 2081 QFRVPGTSTS ATLTGLTRGA
TYNVIVEALK DQQRHKVREE 2121 VVTVGNSVNE GLNQPTDDSC FDPYTVSHYA
VGDEWERMSE 2161 SGFKLLCQCL GFGSGHFRCD SSRWCHDNGV NYKIGEKWDR 2201
QGENGQMMSC TCLGNGKGEF KCDPHEATCY DDGKTYHVGE 2241 QWQKEYLGAI
CSCTCFGGQR GWRCDNCRRP GGEPSPEGTT 2281 GQSYNQYSQR YHQRTNTNVN
CPIECFMPLD VQADREDSRE
[0152] The selected Selective Binding Domain can be fused, attached
or associated with a Tissue Factor polypeptide to generate The
Selective Tissue Vascular Thrombogen by any available procedure.
For example, the Tissue Factor extracellular domain (e.g., SEQ ID
NO: 3, 4, 5 or 6) can be made by known procedures. Such a Tissue
Factor polypeptide can be modified to contain a convenient
attachment site or moiety at any location that does not
substantially interfere with initiation of thrombosis. One
convenient attachment site is the N-terminus of the Tissue Factor
polypeptide.
[0153] In one embodiment, the Selective Binding Domain is fused to
the Tissue Factor polypeptide by use of recombinant technology. One
of skill in the art can readily employ known cloning procedures to
fuse a nucleic acid encoding the desired Selective Binding Domain
to a nucleic acid encoding a Tissue Factor polypeptide. See, e.g.,
Sambrook et al., Molecular Cloning, A Laboratory Manual, Cold
Spring Harbor Laboratory, N.Y., 1989; Sambrook et al., Molecular
Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory, N.Y.,
2001.
[0154] The selected Selective Binding Domain can also be attached
or associated to a Tissue Factor polypeptide by formation of
covalent or non-covalent bonds. Such attachment or association can
be done directly or indirectly to the N-terminus, or other
convenient site, on the Tissue Factor polypeptide. For example, an
indirect attachment or association can be achieved via a convenient
reactive moiety or through a flexible linker to facilitate
formation of the Selective Tissue Vascular Thrombogen and/or to
promote proper association between the Tissue Factor polypeptide
and the cellular membrane to which it will associate. Use of such a
linker to achieve optimal integration and functioning of the
domains within the Selective Tissue Vascular Thrombogen is at the
discretion of one of skill in the art, who can readily ascertain
whether membrane association by the Tissue Factor-Selective Binding
Domain protein is improved by use of a linker.
[0155] By way of example, attachment of a Selective Binding Domain
can be at the N-terminus of a Tissue Factor polypeptide that has
been modified to contain a reactive moiety, for example a cysteine,
at the N-terminus. A cysteine can be attached to the N-terminus of
Tissue Factor, for example, by attaching a peptide containing poly
His tag and a processing protease (FXa) cleavage site followed by a
cysteine (MXXX-HHHHHH-XXXX-IEGR-C, SEQ ID NO: 18) to the N-terminus
of Tissue Factor (SEQ ID NO: 6). Factor Xa digestion cleaves off
the majority of this peptide but leaves a cysteine at the
N-terminus of the Tissue Factor polypeptide. This Cys-Tissue Factor
polypeptide can then be attached to a desired Selective Binding
Domain by available protein ligation reactions (see, e.g.,
Erlanson, Chytil et al. 1996).
[0156] A Selective Binding Domain comprising a compound, peptide or
polypeptide can therefore be linked to the N-terminus of the Tissue
Factor extracellular domain (SEQ ID NO: 3, 4, 5 or 6) through a
disulfide bond. Alternatively, such a Selective Binding Domain can
be linked to a lysine containing linker and then attached to a
Cys-Tissue Factor polypeptide by a thiazolidine ring formed by
reaction the cysteine and the lysine (see, e.g., Zhang, Torgerson
et al. 1998). One such lysine containing linker is KSGGG (SEQ ID
NO: 19). In one embodiment, the D-.beta.-E dipeptide is attached to
the C-terminal glycine of SEQ ID NO: 19 and this seven-amino acid
peptide is linked to an N-terminal cysteine of a Tissue Factor
polypeptide via a thiazolidine ring formed by reaction the cysteine
and the lysine.
[0157] In another embodiment, a biotin and streptavidin can be used
to associate a Selective Binding Domain with a Tissue Factor
polypeptide. Because the binding of biotin to streptavidin is so
stable, there is no need for covalent linkage. Instead, biotin can
be linked to a Selective Binding Domain and Streptavidin can be
linked to a selected Tissue Factor polypeptide. The two
preparations can be incubated together to form a Selective Binding
Domain-biotin:streptavidin-Tissue Factor complex that is a
functional Selective Tissue Vascular Thrombogen. Of course, one of
skill in the art can, alternatively, link streptavidin to a
Selective Binding Domain and biotin to a Tissue Factor polypeptide
to achieve a similar complex.
[0158] One such biotin:streptavidin Selective Tissue Vascular
Thrombogen was made and tested for thrombogenic activity. This
complex had a biotinylated PSMA inhibitor,
Asp-.beta.-linked-L-Glutamate (D.beta.E ), as a Selective Binding
Domain. A streptavidin moiety was N-terminally attached to the
extracellular domain of Tissue Factor (SEQ ID NO: 5). After
incubation of the two domains a D.beta.E-biotin:streptavidin-Tissue
Factor complex was formed. Injection of this complex into animals
leads to extensive thrombosis and necrosis of tumors within the
animals.
[0159] Before use as a therapeutic agent, the Selective Tissue
Vascular Thrombogens can be mixed with Factor VIIa under conditions
permitting formation of the functional Selective Tissue Vascular
Thrombogen:Factor VIIa thrombogenic complex.
[0160] Specific Selective Tissue Vascular Thrombogens
[0161] In one embodiment, the Selective Tissue Vascular Thrombogen
has a Selective Binding Domain that is an integrin binding site
comprising the Fibronectin type III repeat domains 8-11 from human
fibronectin. One example of this type of Selective Tissue Vascular
Thrombogen has SEQ ID NO: 9:
11 1 MRGSHHHHHH GSGSSTPPPT DLRFTNIGPD TMRVTWAPPP 41 SIDLTNFLVR
YSPVKNEEDV AELSISPSDN AVVLTNLLPG 81 TEYVVSVSSV YEQHESTPLR
GRQKTGLDSP TGIDFSDITA 121 NSFTVHWIAP RATITGYRIR HHPEHFSGRP
REDRVPHSRN 161 SITLTNLTPG TEYVVSIVAL NGREESPLLI GQQSTVSDVP 201
RDLEVVAATP TSLLISWDAP AVTVRYYRIT YGETGGNSPV 241 QEFTVPGSKS
TATISGLKPG VDYTITVYAV TGRGDSPASS 281 KPISINYRTE IDKPSQMQVT
DVQDNSISVK WLPSSSPVTG 321 YRVTTTPKNG PGPTKTKTAG PDQTEMTIEG
LQPTVEYVVS 361 VYAQNPSGES QPLVQTAVTS SSGTTNTVAA YNLTWKSTNF 401
KTILEWEPKP VNQVYTVQIS TKSGDWKSKC FYTTDTECDL 441 TDEIVKDVKQ
TYLARVFSYP AGNVESTGSA GEPLYENSPE 481 FTPYLETNLG QPTIQSFEQV
GTKVNVTVED ERTLVRRNNT 521 FLSLRDVFGK DLIYTLYYWK SSSSGKKTAK
TNTNEFLIDV 561 DKCENYCFSV QAVIPSRTVN RKSTDSPVEC MGQEKGEFR
[0162] In another embodiment, the Selective Tissue Vascular
Thrombogen has a Selective Binding Domain that is an integrin
binding site from the Fibronectin 10.sup.th type III repeat domain
of human fibronectin fused to an extracellular domain of Tissue
Factor. An example of this type of Selective Tissue Vascular
Thrombogen has SEQ ID NO: 10:
12 1 MRGSHHHHHH GSGSSTVSDV PRDLEVVAAT PTSLLISWDA 41 PAVTVRYYRI
TYGETGGNSP VQEFTVPGSK STATISGLKP 81 GVDYTITVYA VTGRGDSPAS
SKPISINYRT SSSGTTNTVA 121 AYNLTWKSTN FKTILEWEPK PVNQVYTVQI
STKSGDWKSK 161 CFYTTDTECD LTDEIVKDVK QTYLARVFSY PAGNVESTGS 201
AGEPLYENSP EFTPYLETNL GQPTIQSFEQ VGTKVNVTVE 241 DERTLVRRNN
TFLSLRDVFG KDLIYTLYYW KSSSSGKKTA 281 KTNTNEFLID VDKGENYCFS
VQAVIPSRTV NRKSTDSPVE 321 CMGQEKGEFR
[0163] Methods of Use
[0164] The invention provides a method of treating a solid tumor in
an animal that includes administering a therapeutically effective
amount of a Selective Tissue Vascular Thrombogen of the invention
to the animal. Such a Selective Tissue Vascular Thrombogen has at
least one Selective Binding Domain associated with one or more
thrombogenically active Tissue Factor polypeptides. Additional
domains can be added to achieve optimized localization and/or
thrombogenic activity. As described above, the Selective Binding
Domain can selectively bind to a blood channel within a tumor and
the Tissue Factor Domain can induce localized thrombin production
and thrombosis within the blood channel. According to the
invention, such thrombosis results in tumor infarction and
necrosis. In a preferred embodiment, the Selective Tissue Vascular
Thrombogen, and compositions of the invention are administered
intravenously in solution or, alternatively, in liposomes.
[0165] In another embodiment, the invention provides methods of
inhibiting tumor vascularization by administering PSMA inhibitors.
According to the invention, PSMA can influence vascularization of
prostate tumors. Moreover, inhibitors of PSMA activity can exert a
cytotoxic effect on prostate tumor cells that express PSMA. PSMA
inhibitors can also have a synergistically beneficial effect when
administered with other chemotherapeutic agents and with the
Selective Tissue Vascular Thrombogens of the invention.
[0166] The balance between gamma-glutamate hydrolase and synthase
activity is known to effect cancer cell susceptibility to
anti-folate chemotherapy. Over-expression of gamma glutamyl
hydroxylase activity, which is an activity of PSMA, can promote to
cancer cell resistance to anti-folate drugs (Rhee, Wang et al.
1993). Methotrexate is the most widely used anti-folate for
clinical cancer chemotherapy; its own retention in cell is also
dependent on polyglutamation. PSMA can remove glutamate from folic
acid and other cellular components. Prostate cancers are
notoriously resistant to chemotherapy, possibly because PSMA is
over-expressed in prostate tumor cells.
[0167] However, according to the invention, PSMA inhibitors have a
synergistic effect on reducing cancer cell growth when combined
with anti-folate chemotherapeutic agents and PSMA inhibition can
enhance the sensitivity of prostate cancer to anti-folate drugs.
Accordingly, PSMA inhibitors can be administered with other
chemotherapeutic agent such as an anti-folate drug used to treat
prostate cancer. Such anti-folate drugs include those listed herein
(Table 3). PSMA inhibitors include any inhibitor available to one
of skill in the art that inhibits the activity of PSMA, for
example, any inhibitor of the gamma glutamyl hydrolase activity of
PSMA. PSMA inhibitors contemplated by the invention include listed
herein, especially, the Asp-.beta. linked-Glu dipeptide,
N-succinyl-glutamic acid, quisqalic acid (Sigma),
2-(phosphonomethyl)pent- anedioic acid and related compounds.
Quisqalic acid is a non-competitive inhibitor of NAALADase activity
with K.sub.i=1.9 uM, and D-.beta.-E is a competitive inhibitor with
K.sup.i=0.7 uM. PSMA enzymatic activity can substantially reduced
with such inhibitors.
[0168] Any solid tumor can be treated by the present methods. For
example, the solid tumor can be any of the tumors or carcinomas
listed herein. Examples of tumors and carcinomas contemplated
include lung, breast, ovary, stomach, pancreas, larynx, esophagus,
testes, liver, parotid, biliary tract, colon, rectum, cervix,
uterus, endometrial, kidney, bladder, prostate, thyroid, squamous
cell carcinoma, adenocarcinoma, small cell carcinoma, melanoma,
glioma, or neuroblastoma tumor. In one embodiment the tumor is a
prostate tumor.
[0169] Any chemotherapeutic agent known to one of skill in the art
can also be administered in conjunction with the PSMA inhibitors
and Selective Tissue Vascular Thrombogens of the invention.
According to the invention, combinations of therapeutic agents that
include the present Selective Tissue Vascular Thrombogens can act
synergistically to provide enhanced tumor necrosis.
Chemotherapeutic agents that can be co-administered with these
Thrombogens and inhibitors of the invention include, for example,
methotrexate, doxorubicin, paclitaxil, carboplatin and the like.
Further examples of chemotherapeutic agent that can be administered
with the Selective Tissue Vascular Thrombogens of the invention are
provided in Table 3.
13TABLE 3 Chemotherapeutic Agents Chemotherapeutic Agent Median
Dosage Aldesleukin 22 million units Asparaginase 10,000 units
Bleomycin Sulfate 15 units Carboplatin 50-450 mg Carmustine 100 mg
Cisplatin 10-50 mg Cladribine 10 mg Cyclophosphamide (lyophulized)
100 mg-2 gm Cyclophosphamide (non-lyophilized) 100 mg-2 gm
Cytarabine (lyophilized powder) 100 mg-2 gm Dacarbazine 100 mg-200
mg Dactinomycin 0.5 mg Daunorubicin 20 mg Diethyistilbestrol 250 mg
Doxorubicin 10-150 mg Epoetin Alfa 2,000-10,000 units Etidronate
300 mg Etoposide 100 mg Filgrastim 300-480 mcgm Floxuridine 500 mg
Fludarabine Phosphate 50 mg Fluorouracil 500 mg-5 gm Goserelin 3.6
mg Granisetron Hydrochloride 1 mg Idarubicin 5-10 mg Ifosfamide 1-3
gm Immune Globulin 500 mg-10 gm Interferon Alpha-2a 3-36 million
units Interferon Alpha-2b 3-50 million units Leucovorin Calcium
50-350 mg Leuprolide 3.75-7.5 mg Levamisole 50 mg Mechiorethamine
10 mg Medroxyprogesterone 1 gm Melphalan 50 gm Methotrexate 20 mg-1
gm Mitomycin 5-40 mg Mitoxantrone 20-30 mg Octreotide 1,000-5,000
mcgm Ondansetron Hydrochloride 40 mg Paclitaxel 30 mg Pamidronate
Disodium 30-*90 mg Pegaspargase 750 units Plicamycin 2,500 mcgm
Sargramostim 250-500 mcgm Streptozocin 1 gm Thiotepa 15 mg
Teniposide 50 mg Vinblastine 10 mg Vincristine 1-5 mg
[0170] The activity and pharmacological effects of the Selective
Tissue Vascular Thrombogens and inhibitors of the invention can be
characterized using any method available to one of skill in the
art. In one embodiment, these Selective Tissue Vascular Thrombogens
and inhibitors can be tested in vivo using prostate cancer model
animals, for example, in Lucap58, Mat Lu and LnCaP tumors.
Therapeutic regimens and dosages can also be optimized by observing
the degree of in vivo infarction of Lucap58, Mat Lu and LnCaP
tumors after administration of compositions contained the present
Selective Tissue Vascular Thrombogens and/or inhibitors. Inhibition
of tumor growth can also be used to determine ED.sub.50 (median
effective dose) of the Selective Tissue Vascular Thrombogens and
PSMA inhibitors. The activity and pharmacological effects of the
Selective Tissue Vascular Thrombogens and inhibitors of the
invention can also be characterized in vitro using tumors and tumor
cells in culture.
[0171] For example, compositions containing inhibitors and/or
Selective Tissue Vascular Thrombogens can be analyzed for
efficiency of induction of apoptosis, for example, by measuring
apoptosis in prostate cancer cells and endothelial cells using a
TUNEL assay (Boehringer Mannheim). The efficacy of the inhibitors
can be determined using a calorimetric cell proliferation assay
(Boehringer Mannheim), which is based on the cleavage of
tetrazolium salt WST-1 by mitochondrial dehydrogenases in viable
LnCap cells and other PSMA positive cells. PSMA inhibitors can
further be characterized in vitro by enzymatic assay of PSMA gamma
glutamyl hydrolase activity in the presence and absence of selected
PSMA inhibitors. For example, the ability of an inhibitor to
inhibit PSMA activity can be assessed using a .gamma.-glutamyl
hydrolase assay with 4-NH2-10CH.sub.3 PteGlu.sub.5 as a substrate
(O'Connor, Rotundo et al. 1991; Wang, Rotundo et al. 1993). The Ki
for inhibitors tested in this assay can be determined to serve as a
reference point in determining the proper in vitro and in vivo
dosages for that inhibitor.
[0172] Compositions
[0173] The Selective Tissue Vascular Thrombogens and inhibitors of
the invention can be formulated as pharmaceutical compositions and
administered to a mammalian host, such as a human patient in a
variety of forms adapted to the chosen route of administration.
Preferred routes for administration include, for example,
intravenous and intraarterial routes.
[0174] Solutions of the active constructs and inhibitors or their
salts can be prepared in water or saline, and optionally mixed with
a nontoxic surfactant. Formulations for intravenous or
intraarterial administration may include sterile aqueous solutions
that may also contain buffers, liposomes, diluents and other
suitable additives.
[0175] The pharmaceutical dosage forms suitable for injection or
infusion can include sterile aqueous solutions or dispersions
comprising the active ingredient that are adapted for
administration by encapsulation in liposomes. In all cases, the
ultimate dosage form must be sterile, fluid and stable under the
conditions of manufacture and storage.
[0176] Sterile injectable solutions are prepared by incorporating
the active constructs and inhibitors in the required amount in the
appropriate solvent with various of the other ingredients
enumerated above, as required, followed by filter
sterilization.
[0177] Useful dosages of the constructs and inhibitors can be
determined by comparing their in vitro activity, and in vivo
activity in animal models. Methods for the extrapolation of
effective dosages in mice, and other animals, to humans are known
to the art; for example, see U.S. Pat. No. 4,938,949.
[0178] In general, a suitable dose will be in the range of from
about 1 to about 2000 .mu.g/kg, for example, from about 2.0 to
about 1500 .mu.g/kg of body weight per treatment. Preferred doses
are in the range of about 3 to about 500 .mu.g per kilogram body
weight of the recipient per treatment, more preferably in the range
of about 10 to about 300 .mu.g/kg/treatment, most preferably in the
range of about 20 to about 200 .mu.g/kg/treatment.
[0179] The compound is conveniently administered in unit dosage
form; for example, containing 5 to 1000 .mu.g, conveniently 10 to
750 .mu.g, most conveniently, 50 to 500 .mu.g of active ingredient
per unit dosage form.
[0180] Ideally, the active ingredient should be administered to
achieve peak plasma concentrations of the active compound of from
about 0.1 to about 10 nM, preferably, about 0.2 to 10 nM, most
preferably, about 0.5 to about 5 nM. This may be achieved, for
example, by the intravenous injection of a 0.05 to 25% solution of
the active ingredient, optionally in saline. Desirable blood levels
may be maintained by continuous infusion to provide about 0.01-10.0
.mu.g/kg/hr or by intermittent infusions containing about 0.4-50
.mu.g/kg of the active ingredient(s).
[0181] The desired dose may conveniently be presented in a single
dose or as divided doses administered at appropriate intervals, for
example, as two, three, four or more sub-doses per day. The
sub-dose itself may be further divided, for example, into a number
of discrete loosely spaced administrations; such as multiple
intravenous doses. For example, it is desirable to administer the
present compositions intravenously over an extended period, either
by continuous infusion or in separate doses.
[0182] The ability of the constructs and inhibitors of the
invention to act as thrombosis-inducing agents and tumor inhibitors
may be determined using pharmacological models known to the art, or
using tests described herein.
[0183] The invention will be further described by reference to the
following detailed examples, which are given for illustration of
the invention, and are not intended to be limiting thereof.
EXAMPLE 1
[0184] Activation of Coagulation by a Fibronectin-Tissue Factor
Selective Tissue Vascular Thrombogen
[0185] In this example, a recombinant protein that contains the
N-terminal, extracellular domain of Tissue Factor was fused to the
integrin-binding domain of fibronectin type III repeat domains 8-11
(SEQ ID NO: 9). As illustrated below, this Selective Tissue
Vascular Thrombogen conferred coagulation activity to
integrin-expressing cells that otherwise did not activate the
coagulation cascade. These data showed that the coagulation cascade
is efficiently activated by creation of a four domain protein
incorporating the extracellular domain of Tissue Factor and the
fibronectin type III repeat domains 8-11. This Selective Tissue
Vascular Thrombogen became associated with the cellular membrane
carrying an integrin polypeptide. According to the invention, this
paradigm can be used to engineer Tissue Factor-based thrombogens
that are capable of occluding the blood vessels in a
tissue-selective and/or cell-selective manner.
[0186] Construction of a Fibronectin-Tissue Factor Fusion
Protein
[0187] Fibronectin nucleic acids were obtained by PCR amplification
from marathon-ready cDNA library of human placental origin
(Clontech, Inc.) using vent DNA polymerase (New England Biolabs)
and the following primers:
14 5' CACCAACAACTTGCATCTGGAGGC 3' and (SEQ ID NO:11) 5'
AACATTGGGTGGTGTCCACTGGGC 3' (SEQ ID NO:12)
[0188] After 35 cycles of 1 min 94.degree. C., 1 min 60.degree. C.,
and 1 min 75.degree. C., a 1445 bp fragment was purified. The 1445
fragment was used as template for another PCR amplification using
the following primers:
[0189] 5' ACCATCACGGATCCGGGGTCGTCGACACCTCC TCCCACTGACCTGCGA 3' (SEQ
ID NO: 13, the "FN5a primer") and 5' GGTACC
GGAGGAGCTCGTTACCTGCAGTCTGAACCAGAGG 3' (SEQ ID NO: 14). An 1131 bp
fragment was obtained.
[0190] Tissue Factor nucleic acids were obtained by amplification
from plasmid pTrcHisC-tTF (Stone et. al. 1995) using the following
primers:
[0191] 5' ACGAGCTCCTCCGGTACCACAAATACTGTGGGCAGC 3' (SEQ ID NO: 15
and 5' TCTGCGTTCTGATTTAATCT 3' (SEQ ID NO: 16, the "ptrc-seg"
primer) to produce a 714 bp fragment.
[0192] The 1131 bp fragment and 714 bp fragment were combined and
amplified as a fusion by PCR with the FN5a (SEQ ID NO: 13) and
ptrc-seq (SEQ ID NO: 16) primers to yield a 1827 bp fragment. This
1827 fragment was digested with HindIII, partially digested with
BamHI, and the resulting 1753 bp fragment was ligated into the
BamHI and HindIII sites of the vectorpTrcHisC (Invitrogen).
[0193] The resulting plasmid (FNTF2) encodes a protein fusion
having SEQ ID NO: 9 with a short His 6 Tag at the N-terminus,
followed by fibronectin residues 1237 to 1603, a five-residue
linker peptide, and Tissue Factor residues 3-218 at the C-terminus.
Plasmid FNTF2 was transformed into the E. coli host BL21
(Stratagene) for protein production.
[0194] Proteins
[0195] The soluble extracellular domain of Tissue Factor (SEQ ID
NO: 5, termed TF3-218) was expressed in E. coli, then purified and
refolded as previously described (Stone, Ruf et al. 1995). Factor X
was purified from plasma (Fair, Plow et al. 1979), followed by
immunoaffinity chromatography with immobilized monoclonal antibody
F21-4.2 to reduce VII contamination (Dickinson, Kelly et al. 1996).
Factor VII was affinity purified with a calcium dependent antibody
to the Gla domain and followed by a Mono-Q ion-exchange
chromatography that is associated with spontaneous activation of
VII to VIIa.
[0196] The fibronectin-Tissue Factor (Fn-TF) fusion protein was
expressed in E. coli. and refolded as follows. Briefly, BL21
bacteria were pelleted from cultures obtained 5 hours after IPTG
induction. Bacteria were lysed using lysozyme digestion. Inclusion
bodies were isolated using repeated sonication and centrifugation,
then resuspended in Ni-NTA affinity purification buffer containing
6M guanidium chloride by sonication. The suspension was affinity
purified using Ni-NTA column. Purified fractions were combined and
DTT was added to final concentration of 50 mM at room temperature
overnight to reduce disulfide bonds. Refolding of the protein was
at 4.degree. C. for 4 days in buffer containing 50 mM Tris, 2M urea
and a combination of oxidized glutathione (0.5 mM) and reduced
glutathione (2.5 mM). The refolded soluble fraction was collected
and cleaned with a round of size-exclusion chromatography. The
purified fusion protein appears as a homogenous band of
approximately 96 kD on a silver staining gel (FIG. 2A) that reacts
positively with anti-Tissue Factor antibodies (FIG. 2B) upon
Western blot analysis.
[0197] The LD.sub.50 (median lethal dose) of wild type soluble TF
(SEQ ID NO: 4) in 20 gm Balb/C mice is greater than 500 ug, while
the LD.sub.50 of the Fn-TF construct was about 8 ug.
[0198] Western Blot Analysis
[0199] The immunoreactivity of Tissue Factor was quantified by
Western blot with an anti-human Tissue Factor (anti-huTF) antibody.
Varying amounts of protein were electrophoretically separated and
transferred to nitrocellulose membrane. Membranes were blocked with
5% non-fat milk in TBS. Primary antibody, at a concentration of 1
ug/ml, was incubated with the membranes for 1 hour at 37.degree. C.
An appropriate enzyme-linked secondary antibody was used to permit
visualization of Tissue Factor bands using an enhanced
chemiluminescence system (Amershan-Pharmacia). The intensity of the
bands are quantified with scanning laser densitometry and compared
to that of Tissue Factor standards of known concentration.
[0200] Amidolytic Assay of Bound Factor VIIa to Fn-TF or TF
1-218.
[0201] The catalytic activity of Factor VIIa bound to Fn-TF for a
peptidyl substrate was analyzed by hydrolysis of chromozym t-Pa
(Boehringer Mannheim) and compared with that of the soluble TF
1-218. Varying concentrations TF 1-218 or Fn-TF were incubated with
Factor VIIa at a final concentration of 5nM in the presence of 5 mM
Ca.sup.++ at ambient temperature for 15 minutes. Chromozym t-PA was
added to a concentration of 1 mM. The initial rate of hydrolysis
was measured at 406 nm with a kinetic micro-titer plate reader for
1 minute.
[0202] Proteolytic Activity of Tissue Factor Constructs Toward
Factor X
[0203] The proteolytic activities of both Fn-TF:Factor VIIa and TF
1-218:Factor VIIa complexes toward Factor X were determined by a
functional assay (Schullek, Ruf et al. 1994) using Spectrozyme
Factor Xa to assess Factor Xa generation. Briefly, varying
concentrations of Fn-TF and TF1-218 were pre-incubated with Factor
VIIa (75 nM) for 5 minutes at 37.degree. C. in the presence of 5 mM
CaCl.sub.2. The reaction was initiated by addition of substrate
Factor X (1.5 uM). After incubation for 10 minutes at 37.degree.
C., the reaction was terminated by adding EDTA to a concentration
of 0.1M. The amount of Factor Xa generated was determined by
measuring Factor Xa amidolytic activity using 50 mM of the
chromogenic substrate Spectrozyme Fxa (American Diagnostica,
Greenwich, Conn.). The rate of absorbance increase at 405 nM was
measured in a kinetic micro-titer plate reader.
[0204] Coagulation Assay
[0205] Coagulation assays were performed using an established
procedure with some modifications to include a step for binding
Tissue Factor constructs to cells. Platelet depleted, citrated
human plasma pooled from multiple donors was used for these
experiments. Cells are dislodged with trypsin free cell
dissociation buffer (Gibco), and washed twice with TBS. Cells were
then counted with cytometer. Only cells with viability greater than
90% were used for the assay. Varying concentrations of thrombogen
were incubated with 10.sup.5 cells in 100 ul TBS containing
Ca.sup.++ 10 mM and Mg.sup.++ 5 mM for 15 minutes at 37.degree. C.
The assays were initiated by addition of 100 ul of pooled citrated
plasma pre-warmed to 37.degree. C. Clotting times were recorded as
the interval between assay initiation and clot appearance.
[0206] Model Ternary Structure of Tissue Factor Constructs with
Factor VIIa:Factor X
[0207] The model of ternary structure is based on the crystal
structure of TF and Factor VIIA (Banner, D'Arcy et al. 1996). A Gla
deleted Factor X structure (Padmanabhan et al. 1993) is the primary
source for the Factor X model docked onto the Tissue Factor:VIIa
complex using the InsightII program docking module.
[0208] Results
[0209] Production of a Fibronectin-Tissue Factor Fusion Protein
[0210] Two fibronectin-TF fusion proteins were created by
recombinant methods described above to test the feasibility of
localizing the thrombogenic activity of Tissue Factor selectively
to the cell surface of integrin-expressing cells. The human
fibronectin sequence that encodes typeII repeats 8 through 11 was
amplified by PCR and fused to sequences encoding the extracellular
segment of TF residues 3-218 to generate a protein with SEQ ID NO:
9. Another fusion protein having SEQ ID NO: 10 contained the
fibronectin type III repeat domain 10 with the TF3-218 polypeptide.
These proteins had similar properties and are referred to herein as
Fn-TF or Tn-TF proteins. Like TF 1-218, when expressed in E. coli,
the Fn-TF proteins accumulated in inclusion bodies. The Fn-TF
proteins also behaved similarly to TF1-218 through refolding and
purification. A silver staining gel of purified TF 1-218 and Fn-TF
(SEQ ID NO: 9) is shown in FIG. 2A, and a Western blot stained with
anti-Tissue Factor antibodies is provided in FIG. 2B.
[0211] Amidolytic Activity of Factor VIIa Bound to Fn-TF
[0212] The activity of Fn-TF as cofactor for enhancement of Factor
VIIa amidolytic activity is shown in FIG. 3. The binding of Factor
VIIa to Tissue Factor involves extensive regions from both proteins
encompassing a number of amino acid residues and forming a large
interacting surface. The N-terminal portion of Tissue Factor is
associated with the protease domain of Factor VIIa through residues
Phe76, Tyr94, and Trp45. These interactions are considered
important for allosteric activation of Factor VIIa activity.
[0213] Very little difference in amidolytic activity is discernable
for Tissue Factor and Fn-TF except at high concentrations. This
suggests the that affinity of Tissue Factor for Factor VIIa is not
affected by the addition of fibronectin domains in the FN-TF fusion
protein. Furthermore, the addition of fibronectin moiety to the
N-terminus of Tissue Factor does not affect the subtle
protein-protein interactions in the protease domain of Factor VIIa
that are responsible for the allosteric enhancement of Factor VIIa
amidolytic activity.
[0214] Proteolytic Activity of Fn-TF:Factor VIIa Toward Factor
X
[0215] The effect of the fused docking structure on the proteolytic
activity of TF:VIIa was studied by a linked functional assay
(Schullek, Ruf et al. 1994). Varying concentrations of Fn-TF or
TF1-218 were allowed to bind to 10 pM Factor VIIa for 5 minutes. To
measure the proteolytic activity of the resulting TF:Factor VIIa or
FN-TF:Factor VIIa complexes, Factor X was added to a concentration
of 100 nM and the mixture was incubated for 10 minutes at 37
.degree. C. The reaction was stopped by addition of 50 mM EDTA and
the amount of Factor Xa generated was determined by measuring the
Factor Xa amidolytic activity in the assay mixture using spetrozyme
FXa as described above.
[0216] FIG. 4 shows the proteolytic activity of both soluble
Fn-TF:Factor VIIa and Tissue Factor:Factor VIIa complexes toward
Factor X. The increasing concentration of Fn-TF led to increased
proteolytic activity similar to the curve obtained with TF1-218.
These data suggest that the fused docking structure does not
interfere with the recognition of Factor X by Fn-TF:Factor VIIa
complex. Residues K165 and K166 in Tissue Factor are thought to be
involved in binding to Factor X. When these residue are changed to
alanine, there is no effect on the amidolytic activity of TF:VIIa
activity, but there is a noted change in its proteolytic activity,
thus indicating these residue are important in orienting the Factor
X to allow the most efficient processing of the factor. The
proteolytic activity of the protease complex is greatly enhanced
when the Tissue Factor:Factor VIIa complex is properly docked onto
an anionic lipid surface. This increase in activity may be
explained by the interaction of the anionic lipid surface with the
Gla domain of Factor VIIa bound to Tissue Factor, which will
physically align the complex with substrate Factor X. The
interaction of the Gla domain of both Factor VIIa and Factor X with
phospholipids properly orients Factor X in relation to Factor VIIa
and Tissue Factor. Proper orientation of the various proteins
increases the recognition of Factor X by the Tissue Factor:Factor
VIIa complex and promotes the activation of Factor X to Factor
Xa.
[0217] Binding of Fn-TF to Integrin-Expressing CHO Cells
[0218] The beta-1 family (VLA) of integrins is widely distributed
on the CHO cells. No endogenous Tissue Factor expression in CHO K1
cells was detected by Western Blot analysis or by the coagulation
assay (data not shown). Hence, CHO K1 cells are an ideal system to
study the docking of Fn-TF to integrin.
[0219] The binding of the Fn-TF protein to the integrin on CHO
cells was monitored through direct incubation of varying
concentrations of recombinant Fn-TF with 10.sup.5 CHO K1 cells in
TBS, 10 mM CaCl.sub.2, 5 mM MgSO.sub.4. The bound and unbound
proteins were separated by centrifugation, and the bound Tissue
Factor immunoreactivity was detected by Western blot. The amount of
Tissue Factor bound was quantified by comparing the intensity of
resulting bands on the membrane to that of a standard curve of
Tissue Factor protein of known concentration using a
densitometer.
[0220] FIG. 5 shows that increasing amounts of Fn-TF become
associated with CHO cells as the amount of Fn-TF increases. In
contrast, soluble TF1-218 has no demonstrable association with CHO
cells.
[0221] Induction of Localized Coagulation by Docked Fn-TF
[0222] Activation of the coagulation cascade was measured using a
coagulation assay. Varying concentrations of Fn-TF or Tissue Factor
were incubated with CHO K1 cells for 15 minutes. Coagulation assays
were initiated with addition of 100 ul of pooled normal human
plasma. The coagulation time was recorded as the interim between
the initiation of assay and the appearance of the first fibrin
strands.
[0223] FIG. 6 shows that the coagulation cascade was efficiently
initiated by the Fn-TF complex bound to CHO K1 cells. In
particular, the coagulation time decreases as the Fn-TF
concentration increases suggesting the fibronectin docking domain
can efficiently bind to the integrin on the CHO cell surface, and
that such a docked Fn-TF complex can adopt the necessary
conformation for initiating the coagulation cascade (FIGS. 6 and
7). With CHO K1 cells alone, plasma coagulation takes greater than
400 seconds (1 milliunit activity). However, when the Fn-TF fusion
protein is bound to form approximately 100,000 Fn-TF:integrin
complexes per cell, the coagulation time is reduced to about 20
seconds (40,000 milliunits activity). The Fn-TF fusion protein
appears to be a more efficient activator of the coagulation cascade
than is achieved with antibody mediated targeting of Tissue Factor
observed previously (Huang, Molema et al. 1997).
[0224] Inhibition of Fn-TF Coagulation Activity by the RGD
Peptide
[0225] The coagulation activity of the Fn-TF fusion protein can be
fully blocked by addition of the RGDS (SEQ ID NO: 17) (FIG. 7).
These data indicate that the RGDS peptide, which binds to the
integrin, inhibits binding of the Fn-TF fibronectin docking domain
to the integrin on the cell surface. The increased coagulation
activity of Fn-TF relative to TF1-218 is therefore apparently due
to the fibronectin selective binding domain, which binds and
orients the extracellular domain of Tissue Factor into proximity
with anionic phospholipid membrane microdomains and thereby
facilitates the association of Factor VIIa with Tissue Factor and
the cell surface.
[0226] Coagulation Activity of Fn-TF Versus Full Length Tissue
Factor
[0227] The coagulation activity of the Fn-TF fusion protein with
CHO K1 cells was compared with the coagulation activity of a CHO K1
cell line that stably expressed full-length recombinant Tissue
Factor. Approximately, 5.times.10.sup.5 Fn-TF molecules are bound
per cell when 75 nM Fn-TF fusion protein is incubated with 10.sup.5
CHO K1 cells in 100 ul. A similar number of CHO K1 cells that are
expressing approximately 5.times.10.sup.5 native Tissue Factor
molecules have approximately the same level of coagulation activity
as the 10.sup.5 CHO K1 cells exposed to 75 nM Fn-TF (FIG. 7).
[0228] Hence, the thrombogenic activity of Tissue Factor is largely
dependent upon binding to a cellular membrane and upon physical
alignment with the cell surface in a manner that is similar to that
of native Tissue Factor structure. Whereas the lack of a membrane
assembly domain eliminates the major mechanism for proper docking
of the Tissue Factor:Factor VIIa:Factor X complex on the cell
surface, as well as the associated protease activities, the data
provided in this example indicate that the N-terminus of Tissue
Factor tolerates introduction of heterologous selective binding
domains and that those binding domains can facilitate proper cell
membrane association and orientation to restore the protease
activity of Tissue Factor.
[0229] Hence, fusion of tissue-selective binding domain to the
extracellular domain of Tissue Factor can target coagulation within
that selected tissue type.
EXAMPLE 2
[0230] Prostate Tumor Infarction By Tissue Factor:PSMA
Inhibitor
[0231] In this example, intravascular thrombosis was induced within
mouse tumors by administration of an Asp-.beta. linked-Glu
(D.beta.E)-biotin:streptavidin-Tissue Factor complex. The
Asp-.beta. linked-Glu dipeptide is a binding inhibitor of PSMA and,
in this example, acts as a Selective Binding Domain. Use of a small
peptide inhibitor such as D-.beta.-E has benefits over the use of
an anti-PSMA antibody because it is easier to produce, and it is
small so that the thrombogenic potential is maximized, for example,
with small tumor vessels. The Asp-.beta. linked-Glu Selective
Binding Domain directs an associated Tissue Factor polypeptide to
PSMA-expressing cells that line the blood channels of prostate
tumors. After association with PSMA-expressing cells, the Tissue
Factor domain initiates localized thrombosis and infarctive
necrosis of the prostate tumor. This results in tumor regression
without harming the animal host.
[0232] Reagents
[0233] Purified human plasma factor VIIa was from Hematologic
Technologies (Essex Junction, Vt.). Liposome incorporated
doxorubicin (Doxil.TM.) was from ALZA corporation (Mountain View,
Calif.). Streptomyces avidinii was from the ATCC and grown for
isolation of DNA using the QIAmp kit method (QIAGEN, Valencia,
Calif.).
[0234] Antibodies
[0235] Anti-PSMA antibodies (7E11C5) were used to characterize the
Mat Lu rat and LuCap mouse prostate tumor models. Monoclonal
antibodies against mouse CD31 (MEC 13.3) and rat CD31 (TLD-3A 12)
were from PharMingen (La Jolla, Calif.). Biotinylated rat
anti-mouse CD31 antibody and a FITC labeled mouse anti-human CD31
antibody were also purchased from Pharmingen, La Jolla, Calif.
Murine monoclonal antibody J591 specific for the extracellular
domain of PSMA was provided by Dr. N. Bander (School of Medicine,
Cornell University). Biotinylated 7E11C-5 antibody was from Dr. J.
Murphy, Pacific Northwest Cancer Foundation, Seattle, Wash. The
7E11C-5 antibody epitope was mapped to the N-terminal intracellular
portion of human PSMA that is not present in the mouse PSMA
homologue. The anti-CD31 antibodies react with endothelial
cells.
[0236] Streptavidin-Tissue Factor Fusion Protein
[0237] To determine whether a PSMA inhibitor could serve as a
Selective Binding Domain to target Tissue Factor to prostate tumors
and then induce tumor necrosis, an Asp-.beta.-linked-L-Glutamate
(D.beta.E) biotinylated dipeptide was synthesized. This peptide was
made to interact with and bind to a streptavidin moiety that was
N-terminally attached to the extracellular domain of Tissue Factor
(SEQ ID NO: 5), termed streptavidin-TF. The streptavidin-TF fusion
protein was produced in E. coli and folded to generate a tetramer
capable of binding to four biotin molecules. The details involved
in generating this protein are described below.
[0238] Tissue Factor cDNA containing amino acids 3 to 311 was
obtained by PCR of a human cDNA library (Clontech, Palo Alto,
Calif.) with the following primers:
15 (SEQ ID NO:20) BM21: 5'-ACTACAAATACTGTGGCAGCA-3'; and (SEQ ID
NO:21) BM33: 5'-TTTAAGCTTTCACGTGCCCATACACTC- TACCGG-3'.
[0239] The resulting 639 bp fragment was isolated by gel
electrophoresis and subjected to a second PCR with primer BM33 (SEQ
ID NO: 21) and the following primer:
16 BM51: 5'-AAATGGATCCTGGTGCCTAGGGGCCCGGGACTACAAATACTGTGGCAGCA-3'.
(SEQ ID NO:22)
[0240] The resulting 670 bp fragment was digested with BamHI and
HindIII and ligated into the BamHI and HindIII sites of the vector
pTrcHisC (Invitrogen, Carlsbad, Calif.). The BM51 oligo also
encodes a thrombin cleavage site (Val-Pro-Arg-Gly-Ser, SEQ ID NO:
23) for selective proteolytic deletion of the His tag from the
expressed protein. This plasmid (NuV120) was further modified to
contain a linker sequence with three repeats of Gly.sub.4Ser (SEQ
ID NO: 24) between the thrombin cleavage sequences and those of
Tissue Factor. The following overlapping oligos were annealed and
inserted into the BamHI and Aval sites of NuV120:
17 nuv20-1: 5' GATCTTGGTCCCTAGGGGATCCGCAGAACCAATGCCT 3'; (SEQ ID
NO:25) nuv20-2: 5' PO.sub.4-CACTCGCTAAACTTCAGTCAATACCTCT- GGTATACT
3'; (SEQ ID NO:26) nuv20-3: 5'
PO.sub.4-GGTACCGGAGGAGGCGGTTCAGGTGGTGGAGGTTCA 3'; (SEQ ID NO:27)
nuv20-4: 5' PO.sub.4-GGAGGTGGAGGTTCTC 3'; (SEQ ID NO:28) nuv20-5:
5' PO.sub.4-TCTGCGGATCCCCTAGGGACCAA 3'; (SEQ ID NO:29) nuv20-6: 5'
PO.sub.4-AGGTATTGACTGAAGTTTAGCGAGTGAGGCATTGGT 3'; (SEQ ID NO:30)
nuv20-7: 5' PO.sub.4-CCACCTGAACCGCCTCC- TCCGGTACCAGTATACCAG 3';
(SEQ ID NO:31) nuv20-8: 5' CCGGGAGAACCTCCACCTCCTGAACCTCCA 3'. (SEQ
ID NO:32)
[0241] The resulting plasmid (NuV127) encodes a His-tag, a thrombin
cleavage site, three repeats of the spacer Gly.sub.4Ser (SEQ ID NO:
37), and Tissue Factor residues 3 to 211. This vector can be used
to create expression vectors for various Selective Tissue Vascular
Thrombogens by inserting a cDNA sequence encoding the derived amino
acids into unique BamHI and KpnI sites. The streptavidin gene was
amplified by PCR with Pfu polymerase (Stratagene) using the
following oligonucleotides:
18 strep1: 5' ACCACGGTCTCGATTACGGC 3'; (SEQ ID NO:33) and strep2:
5' ACTACTGCTGAACGGCGTCG 3'. (SEQ ID NO:34)
[0242] Such PCR amplification results in a 514 bp fragment. The 514
bp fragment was purified and used as template for a second PCR
amplification, this time with the following oligonucleotides:
19 (SEQ ID NO:35) tl,32 strep3: 5' CACACAGGATCCGCCGCCGAGGCC-
GGCATCAC 3'; and (SEQ ID NO:36) strep4: 5'
CACACAGGTACCCTGCTGAACGGCGTCGAGCG 3'.
[0243] BamHI and KpnI sites, respectively, are underlined in the
above oligonucleotide sequences and extra nucleotides in italics
were added for efficient enzyme digestion. The resulting DNA
fragment of 486 bp was purified, digested with BamHI and KpnI and
cloned into the BamHI and KpnI sites of NuV127. The resulting
plasmid, NuV159, expresses a Streptavidin-Tissue Factor.
[0244] BL21 cells transformed with the NuV159 plasmid were grown in
Super Medium (25 g tryptone, 15 g yeast extract and 5 g NaCl per
liter) supplemented with biotin. Cells were induced with 1 mM IPTG
when the OD.sub.600 reached 0.6 and were cultured for 24 hours at
37.degree. C. The protein (Strep-TF) accumulated in inclusion
bodies, which were isolated as described in Donate et al.,
Dimerization of tissue factor supports solution-phase
autoactivation of factor VII without influencing proteolytic
activation of factor X. Biochemistry, 39: 11467-11476, 2000. The
inclusion bodies were solubilized in 6 M guanidinium hydrochloride
(GuHCl), pH 8.0, and the protein construct was partially purified
in a Ni-NTA (QIAGEN) column equilibrated and washed with 6 M GuHCl
pH 8.0 and eluted with 250 mM imidazole, 6 M GuHCl, pH 8.0. Protein
folding was performed by dilution of GuHCl solubilized Strep-TF in
20 mM Tris, 300 mM NaCl, 0.8 M GuHCl pH 8.0 and glutathione redox
buffer. After 16 hours at 4.degree. C., the sample was concentrated
with a Pellicon XL concentrator (MWCO, 10,000). The His tag is
removed by thrombin digestion and the protein construct purified in
two steps with a Source 15Q 16/10 column followed by a Sephacryl
S-200 gel filtration. FIG. 9A depicts a silver-stained gel
illustrating the purity of the streptavidin-TF protein.
[0245] A D.beta.E-biotin:streptavidin-TF complex was prepared by
mixing excess D.beta.E-biotin (>10 to 1 molar concentration
ratio) with streptavidin-TF protein in saline. The binding of the
D.beta.E-biotin peptide to the streptavidin-TF protein was allowed
to proceed for 30 min. The resulting
D.beta.E-biotin:streptavidin-TF complex was dialyzed against two
changes of saline. FIG. 9B illustrates the activity of the
D-.beta.-E-biotin:streptavidin-Tissue Factor complex in a Factor X
generation assay.
[0246] Before injection into animals, the
D.beta.E-biotin:streptavidin-TF complex was mixed with an equal
molar ratio of Factor VIIa for 10 min. This permitted formation of
the functional D.beta.E-TF:Factor-TF:Factor VIIa thrombogenic
complex.
[0247] Cell Culture
[0248] Cell lines LnCap and the Mat Lu cell were from ATCC. The
LnCap cells were cultivated in RPMI-1640 medium supplemented with
10% fetal calf serum, glutamine (2 mM), HEPES (10 mM), sodium
pyruvate (1 mM) and glucose (4.5 g/L). Mat Lu cells were cultured
in RPMI-1640 with 10% fetal calf serum, glutamine (2 mM), and 250
nM dexamethasone.
[0249] Factor Xa Generation Assay
[0250] Factor Xa generation assays were performed as described in
Ruf, et al. (J. Biol. Chem., 266, pg. 2158-66, 1991) with
modifications to provide for association of the STVT constructs to
PSMA expressing LnCap cells. LnCap cells were plated at
8.times.10.sup.4 per well in 96 well plates and allowed to attach
for 4 hours in the medium described above. Medium was replaced with
HBSA buffer (150 mM NaCl, 5 mM CaCl.sub.2, 0.5% BSA, 20 mM Hepes,
pH 7.4) and serial concentrations of D.beta.E:Strep-TF:VIIa or
Strep-TF:VIIa complex were added to the wells. After incubation for
5 minutes, factor X was then added to a final concentration of 1
.mu.M. After 5 minutes at 37.degree. C. the limited proteolytic
conversion of factor X to factor Xa was arrested with 100 mM EDTA.
Factor Xa amidolytic chromogenic substrate Spectrozyme Xa (American
Diagnostica, Greenwich, Conn.) was added to a final concentration
of 200 .mu.M and substrate hydrolysis was determined kinetically at
OD 405 in a spectrophotometric plate reader (Molecular Devices,
Sunnyvale, Calif.).
[0251] Animal Models
[0252] Cell lines LnCap and the Mat Lu cell were from ATCC. The
PSMA positive human prostate carcinoma (CaP) xenograft, LuCap 58,
was carried as a xenograft in WEHI nude mice (The Scripps Research
Institute Breeding Facility). See Bladou et al., In vitro and in
vivo models developed from human prostatic cancer. Prog. Urol., 7:
384-396, 1997. The tumors were passaged by implantation of .about.2
mm.sup.3 fragments in the subcutaneous tissue of the back of the
mice. The rat Mat Lu prostate carcinoma was carried in male
Copenhagen rats aged 4 to 6 weeks (Harlan Sprague-Dawley,
Germantown, N.Y.) innoculated with 5.times.10.sup.5 Mat Lu cells
per subcutaneous site on the back.
[0253] Treatment of tumors was initiated once tumors reached 200
mm.sup.3 through bolus intravenous injection of the Selective
Tissue Vascular Thrombogen or a control protein (0.1 mg/Kg based on
strep-TF protein) and repeated twice at two-day intervals. For
combination therapies, liposomal doxorubicin (Doxil.TM.) at 2 mg/kg
was separately injected intravenously. Tumor growth and other
physical signs were monitored daily including gross evidence of
tumor necrosis, local tumor ulceration as well as evidence of
toxicity including mobility, response to stimulus, eating, and
weight of each animal. The studies have been reviewed and approved
by the Institutional Animal Care and Use Committee of The Scripps
Research Institute. The work was conducted in the TSRI facilities
which are accredited by the Association for the Assessment and
Accreditation of Laboratory Animal Care. The Scripps Research
Institute maintains an assurance with the Public Health Service, is
registered with the United States Department of Agriculture and is
in compliance with all regulations relating to animal care and
welfare.
[0254] Immunohistochemistry
[0255] Immunohistochemical analysis was performed on formalin fixed
as well as fresh frozen 5 .mu.m tissue sections mounted on
poly-lysine coated slides. For endothelial identification,
biotinylated murine anti-rat CD-31 monoclonal antibody (TLD-3A12)
or biotinylated rat anti-mouse CD-31 monoclonal antibody (MEC 13.3)
were used at 1 .mu.g/ml as first antibody then the reaction was
developed with fluorescein conjugated strepavidin. For
identification of PSMA in frozen sections, reaction with mouse
monoclonal antibody J591 was followed by biotinylated rabbit
anti-mouse IgG and the reaction was visualized with Texas-red
conjugated strepavidin. Staining of PSMA in formalin fixed tissue
was performed with biotinylated 7E11C-5 antibody. The tissue
sections were analyzed with the aid of laser scanning confocal
microscopy (Bio-Rad, Hercules, Calif.).
[0256] Coagulation Assay
[0257] The coagulation activity of the
D.beta.E-biotin:streptavidin-TF:Fac- tor VIIa thrombogenic complex
was assayed using a cell mediated coagulation assay employing CHO
K1 cells that stably express PSMA. Different concentrations of
D.beta.E-biotin:streptavidin-TF:Factor VIIa thrombogenic complex
(in 50 ul) were incubated with 10.sup.5 PSMA expressingCHO K1 cells
(also in 50 ul) at room temperature for 15 min to allow the
D.beta.E-biotin:streptavidin-TF:Factor VIIa thrombogenic complex to
associate with PSMA on the cell surface. At the end of this
incubation, 100 ul citrated pooled human plasma was added and the
assay was initiated by adding 100 ul 20 mM CaCl.sub.2 that had been
pre-warmed to at 37.degree. C. Assays using CHO K1 cells without
PSMA were used as controls. The clotting time was recorded as the
interim between the initiation of the reaction and the occurrence
of the first strands of fibrin gel. This assay was used to guide
the construction of fusion proteins and to quantify the activity of
the D.beta.E-biotin:streptavidin- -TF:Factor VIIa thrombogenic
complex in different preparations.
[0258] Combined Treatment with the
D.beta.E-biotin:streptavidin-TF:Factor VIIa Thrombogenic Complex
and Doxorubicin
[0259] Treatment of tumor-bearing rodents was initiated when tumors
reached 200 mm.sup.3 by bolus i.v. injection. Some test animals
were injected with D.beta.E-biotin:streptavidin-TF:Factor VIIa
thrombogenic complex (0.1 mg/Kg based on streptavidin-TF total
protein) plus doxorubicin (2 mg/kg, 3.5umol/kg). Other test animals
were injected with D.beta.E-biotin:streptavidin-TF:Factor VIIa
thrombogenic complex (0.1 mg/Kg based on streptavidin-TF total
protein) alone. Control animals were injected with doxorubicin (2
mg/kg, 3.5umol/kg). Mock-treated animals received no doxorubicin
and no thrombogenic complex. The treatment is repeated daily. The
doxorubicin dosage is determined according to the MTD (maximum
tolerated dose) published for daily injection for five days, which
between 2.8 umol/kg to 3.6 umol/kg. Tumor growth was monitored
daily through 4 weeks and until death. Tumor size was measured
daily from the day of initial treatment.
[0260] Statistical analysis
[0261] Statistical significance was determined by the two-tailed
Student=s t test, except for statistical significance of survival
curves which utilized the Logrank test using GraphPad Prism version
3.00 (GraphPad Software, San Diego Calif. USA).
[0262] Results
[0263] Immunohistological Analysis
[0264] PSMA was detected on the vessels of a xenograft model of
human prostate tumors (LuCap 58) using the 7E11 antibody, and also
using a biotinylated peptidyl inhibitor (Asp-.beta.-Glu) of PSMA
enzymatic activity. Strong PSMA expression was detected on the
luminal surfaces of the vessels of the PSMA positive human LuCap 58
xenograft grown in nude mice (FIG. 8A and 8B). The epitope
recognized by the murine 7E11C-5 antibody was mapped to the
N-terminal intracellular portion of human PSMA that is not present
in the mouse PSMA homologue. Therefore, the detected PSMA was human
PSMA. This observation indicates that the tumor vasculature present
in this tumor model is of human origin, even though the LuCap model
has been propagated in nude mice for much too long for primary
non-transferred endothelial cells to survive in these tumors. Such
data indicate that the tumors themselves are generating
microvascular lining cells or that human PSMA from the tumor cells
was acquired by transfer to endothelial cells of the mouse that
have grown into the tumor.
[0265] Immunohistochemical analysis of the human LuCap tumors
clearly identified PSMA positive cells that line and thereby
delineate the microscopic channels with structural characteristics
not unlike microvascular channels (FIG. 8A and 8B). PSMA expression
is more intense on the aspect of tumor cell membranes that
constitutes the luminal surface of the channels (FIG. 8B).
[0266] A second piece of evidence indicates that the PSMA-positive
cells lining the vessels of the tumors are not endothelial cells.
Frozen sections of LuCap 58 tumors were immunohistochemically
stained with biotinylated rat anti-mouse CD31 antibody and with an
FITC-labeled mouse anti-human CD31 antibody. While the FITC-labeled
anti-mouse CD31 reacted positively with the tumor vessels lining
cells in these sections (FIG. 8C), the rat anti-human CD31 staining
was negative. Double staining of the LuCap tumor with anti-mouse
CD31 antibody and PSMA specific antibody (FIG. 8C) indicated that
these PSMA-expressing microchannels are distinct and mutually
exclusive of microvascular channels lined by CD31 positive cells.
These data therefore indicate that human endothelial cells do not
exist in the LuCap 58 tumor. In Mat Lu tumors, the anti-PSMA
antibody and anti-CD31 antibody reacted with almost entirely
mutually exclusive cell surfaces, also indicating that the PSMA
positive cells lining blood vascular channels are not endothelial
cells.
[0267] To investigate that whether the PSMA-positive cells that
lined the channels in these tumors were part of the tumor
vasculature, bacteriophage M13 was injected into the blood stream
of the animal as a marker. Tumors were harvested minutes after the
injection and frozen sections were prepared from these samples.
Extensive experience with in vivo phage panning has proven that
phage will remain in the tumor vasculature and can be easily
recognized with anti-phage antibody staining. Double staining with
PSMA antibody and anti-phage antibody revealed that PSMA lined the
channel structures stained by phage and through which blood flows
(FIG. 8D). These data indicate that the channels lined by
PSMA-expressing cells are part of the tumor vasculature (see
schematic diagram provided in FIG. 8E). Cells lining the blood
vessels and in contact with the blood are tumor cells, rather than
endothelial cells. Similar microchannels lined by PSMA positive
cells were also observed in syngeneic rat Mat Lu tumors.
[0268] The STVT Functionally Associates with PSMA Positive
Cells.
[0269] A factor Xa generation assay was used to test whether the
D.beta.E-biotin:streptavidin-TF:Factor VIIa complex could align
properly on an anionic cell membrane surface and properly associate
with factor X substrate that has localized to the same locus. As
illustrated in FIG. 9B, the D.beta.E-biotin:streptavidin-TF:Factor
VIIa complex but not the streptavidin-TF:Factor VIIa complex
functions on LnCap cells to generate Factor Xa in the Factor Xa
generation assay described above. Hence, the
D.beta.E-biotin:streptavidin-TF:Factor VIIa complex can
proteolytically convert factor X to factor Xa while bound to LnCap
cells.
[0270] The factor Xa generation assay requires the functional
assembly of the assembled D.beta.E:Strep-TF:VIIa complex on PSMA
expressing LnCap cells. Unlike most tumor cells, LnCap cells do not
express Tissue Factor as indicated by coagulation assays and
western blot examination (data not shown). LnCap cells also do not
directly form factor Xa from factor X and therefore cannot drive
the thrombogenic cascade (see streptavidin-Tissue Factor control in
FIG. 9B). The dose dependent increase of factor Xa generation by
the D.beta.E: Strep-TF:VIIa complex in the presence of LnCap cells
was striking in comparison to the control LnCap cells treated with
the streptavidin-Tissue Factor molecule that lacked the PSMA
targeting element (FIG. 9B). These data indicate that the
D.beta.E:Strep-TF:VIIa complex functionally assembles on the cell
surface through binding of D.beta.E to PSMA and initiates the
thrombogenic cascade.
[0271] Tumor Necrosis
[0272] Mat Lu tumors were generated by subcutaneous inoculation of
0.5.times.10.sup.6 tumor cells per site in the subcutaneous tissue
of the back of the Copenhagen rats. After 7 days the tumors grew to
an average diameter of 1 cm. Treatment is initiated at this time by
bolus intravenous injection of the
D.beta.E-biotin:streptavidin-TF:Factor VIIa complex at a dose of
0.1 mg streptavidin-TF per Kg body weight. Treatment was repeated
daily for 7 days. Tumor growth was measured daily and graphed. Key
physical signs were monitored, including:
[0273] (a) Tumor necrosis and infarction. Mat Lu tumors are
non-necrotic tumors with fast initial growth but slow progression.
Local ulceration is a important sign of targeted thrombosis;
[0274] (b) Apparent health of each rat; and
[0275] (c) Mobility and response to stimulus.
[0276] The control streptavidin-TF protein was not toxic in rats
over a wide range of concentrations, thereby permitting evaluation
of the potential for selective tumor thrombosis and infarctive
necrosis in tumor bearing rats.
[0277] Intravenous injection of the
D.beta.E-biotin:streptavidin-TF: Factor VIIa complex was associated
with a rapid wave of localized microthrombosis of blood channels
within Mat Lu tumors, leading to infarctive necrosis of Mat Lu
prostate tumors. As shown in FIG. 10A, the treated tumor (left) was
extensively necrotic while the untreated tumor (right) showed
little or no necrosis. The center of the treated tumor was
liquefied upon gross and histological pathological examination,
showing gross signs of ischemic necrosis. In contrast, there was no
micro-thrombosis or necrotic regions in tumors from the control
group (FIG. 10B). Occluded tumor microvessels were widespread in
the experimental group (FIG. 10C), with platelet aggregates, packed
erythrocytes and fibrin (FIG. 10D). The tumor interstitium commonly
contained a few erythrocytes and was infiltrated with inflammatory
cells (FIG. 10D). After the standard three infusions at two-day
interval, tumors showed very extensive necrosis with liquefaction
of the entire central region of the tumors. However, at the growth
edge of tumors from the treated animals, a rim of viable tumor
tissue remained.
[0278] Pathological studies were performed to confirm that
intravenous administration of an
D.beta.E-biotin:streptavidin-TF:Factor VIIa complex induced
selective thrombosis of tumor vasculature in rats bearing Mat Lu
prostate cancers. Signs of tumor vasculature thrombosis were
observed in tumors immediately following treatment. The center of
the treated tumors showed gross signs of ischemic changes. In
Hematoxylin and Eosin stained sections, the number of vessels that
were occluded increased dramatically. After one hour, blood vessel
thrombosis was extensive. Occlusive platelet aggregates were
frequently observed in thrombi as well as red blood cells and
fibrin. By 72 hours and after three treatments, the tumors showed
advanced necrosis. In some tumors, the entire central region was
completely liquified.
[0279] FIG. 11A graphically depicts the retardation of Mat Lu tumor
growth by the D.beta.E-biotin:streptavidin-TF:Factor VIIa complex.
In the saline treated control group (square symbols) rats, the
tumor volume increased progressively and was greater than the
D.beta.E-biotin:streptavidin-TF:Fa- ctor VIIa treated group (J
symbols). The tumor size was measured with a caliper and tumor
volume calculated as D.times.d.sup.2. In some cases, although the
tumor center is necrotic and liquified, the total tumor size
remained unchanged or increased slightly as a result of an
inflammatory response and as some surviving tumor at the periphery
of the tumor continued to grow.
[0280] FIG. 11B graphically illustrates the weight of tumors after
removal. The average tumor weight in the
D.beta.E-biotin:streptavidin-TF: Factor VIIa treated group (grey)
was substantially less than that of the control group (black).
[0281] Combined Therapy with Doxorubicin.
[0282] To address the potential to enhance selective tumor
microvascular thrombosis and infarction of tumors, infusions of
both the D.beta.E-biotin:streptavidin-TF: Factor VIIa construct and
low doses of liposomal doxorubicin (2 mg/Kg) were conducted. Three
infusions of each were administered at two-day intervals as
described above. There was virtually a complete arrest of tumor
growth and gross eradication of tumors in some rats that received
doxorubicin with the D.beta.E-biotin:streptavidin-TF: Factor VIIa
construct (FIG. 12A and 12B). This combination therapy also had a
significant beneficial effect on survival of the tumor bearing
animal hosts (p<0.001, FIG. 12B). The prolongation of survival
of rats treated with the D.beta.E-biotin:strepta- vidin-TF: Factor
VIIa alone was modest, but significant. Therapy with low dose
liposomal doxorubicin alone had no measurable benefit (FIGS. 12A
and 12B).
[0283] FIG. 12 graphically illustrates the synergistic effect of
combined treatment with both the D.beta.E-biotin:streptavidin-TF:
Factor VIIa complex and doxorubicin. After about 5-6 days of
treatment, the tumor volume of rats receiving doxorubicin had
progressively increased (square symbols, FIG. 12A). There was
little difference in tumor volume of between the control and
doxorubicin treatment alone (data not shown). However, animals
receiving combined therapy exhibited substantially no increase in
tumor volume and had significantly smaller tumors than did
doxorubicin-only treated animals (round symbols, FIG. 12A).
Similarly, rats receiving the D.beta.E-biotin:streptavidin-TF:
Factor VIIa complex and doxorubicin (long dashed line, FIG. 12B)
survived substantially longer than rats that that received
doxorubicin alone (solid line, FIG. 12B).
[0284] Therapy with PSMA Inhibitors
[0285] FIG. 13A graphically illustrates that as the concentration
of D.beta.E inhibitor increases (circular symbols), the viability
of PSMA expressing prostate cancer cells declines. A cell
proliferation and viability assay was employed to assess D.beta.E
inhibitor activity using trypan blue staining. LnCap cells
(4.times.10.sup.4 cells/well) were seeded in 96 well plates.
Different concentrations of the D.beta.E inhibitor or the Asp-Glu
(D-E) substrate were added to the media at the concentrations
indicated in FIG. 13A. The % cell viability was determined 48 hours
after treatment as the number of living cells (unstained) divided
by total cells count (stain+unstained cells). Inhibition of the
glutamyl preferring carboxypeptidase activity of PSMA using its
inhibitor Aspartyl-.beta.-linked L glutamate (D-.beta.-E) resulted
in tumor cell death in a dose dependent manner in contrast to its
physiological substrate analogue, Aspartyl-glutamate (D-E).
[0286] FIG. 13B graphically illustrates the synergistic effect of
combining methotrexate (MTX) and the PSMA inhibitor, D-.beta.-E, on
cancer cell viability in vitro. The cytotoxic effect of
methotrexate was assessed with and without the presence of the PSMA
inhibitor (D-.beta.-E) or PSMA substrate (D-E) using a cell
proliferation and viability assay. The cytotoxic effect of
methotrexate was potentiated in the presence of inhibitor at a
concentration of 0.1 uM. The ID.sub.50 of MTX was reduced from
around 10 uM to around 0.5 uM in the presence of the PSMA inhibitor
(D-.beta.-E) (ID.sub.50/ID.sub.50*=20), a twenty-fold enhancement
of the tumoricidal activity.
[0287] These experiments illustrated the therapeutic potential of
reduction of a tumor cell viability, combined with selective tumor
vascular thrombosis in prostate cancer by targeting cells that
express PSMA. The data indicate that PSMA-expressing tumor cells
create vascular channels that are lined by tumor cells. Some tumor
tissue on the periphery of the tumor often escaped thrombosis,
indicating that these tumor cells may survive because they are not
in direct contact with tumor blood vessels. However, these
peripheral tumor cells are more accessible to cytotoxic drugs
delivered by the circulatory system. A combination of therapeutic
agents that includes the present Selective Tissue Vascular
Thrombogens and an anti-tumor drug may effectively eradicate solid
tumors.
[0288] Hence, the compositions and methods of the invention are
peculiarly suited to treat here-to-fore inaccessible tumor cells
within the heart of solid tumors.
[0289] The foregoing specification, including the specific
embodiments and examples, is intended to be illustrative of the
present invention and is not to be taken as limiting. Numerous
other variations and modifications can be effected without
departing from the true spirit and scope of the present
invention.
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Sequence CWU 1
1
36 1 295 PRT Homo sapiens 1 Met Glu Thr Pro Ala Trp Pro Arg Val Pro
Arg Pro Glu Thr Ala Val 1 5 10 15 Ala Arg Thr Leu Leu Leu Gly Trp
Val Phe Ala Gln Val Ala Gly Ala 20 25 30 Ser Gly Thr Thr Asn Thr
Val Ala Ala Tyr Asn Leu Thr Trp Lys Ser 35 40 45 Thr Asn Phe Lys
Thr Ile Leu Glu Trp Glu Pro Lys Pro Val Asn Gln 50 55 60 Val Tyr
Thr Val Gln Ile Ser Thr Lys Ser Gly Asp Trp Lys Ser Lys 65 70 75 80
Cys Phe Tyr Thr Thr Asp Thr Glu Cys Asp Leu Thr Asp Glu Ile Val 85
90 95 Lys Asp Val Lys Gln Thr Tyr Leu Ala Arg Val Phe Ser Tyr Pro
Ala 100 105 110 Gly Asn Val Glu Ser Thr Gly Ser Ala Gly Glu Pro Leu
Tyr Glu Asn 115 120 125 Ser Pro Glu Phe Thr Pro Tyr Leu Glu Thr Asn
Leu Gly Gln Pro Thr 130 135 140 Ile Gln Ser Phe Glu Gln Val Gly Thr
Lys Val Asn Val Thr Val Glu 145 150 155 160 Asp Glu Arg Thr Leu Val
Arg Arg Asn Asn Thr Phe Leu Ser Leu Arg 165 170 175 Asp Val Phe Gly
Lys Asp Leu Ile Tyr Thr Leu Tyr Tyr Trp Lys Ser 180 185 190 Ser Ser
Ser Gly Lys Lys Thr Ala Lys Thr Asn Thr Asn Glu Phe Leu 195 200 205
Ile Asp Val Asp Lys Gly Glu Asn Tyr Cys Phe Ser Val Gln Ala Val 210
215 220 Ile Pro Ser Arg Thr Val Asn Arg Lys Ser Thr Asp Ser Pro Val
Glu 225 230 235 240 Cys Met Gly Gln Glu Lys Gly Glu Phe Arg Glu Ile
Phe Tyr Ile Ile 245 250 255 Gly Ala Val Val Phe Val Val Ile Ile Leu
Val Ile Ile Leu Ala Ile 260 265 270 Ser Leu His Lys Cys Arg Lys Ala
Gly Val Gly Gln Ser Trp Lys Glu 275 280 285 Asn Ser Pro Leu Asn Val
Ser 290 295 2 263 PRT Homo sapiens 2 Ser Gly Thr Thr Asn Thr Val
Ala Ala Tyr Asn Leu Thr Trp Lys Ser 1 5 10 15 Thr Asn Phe Lys Thr
Ile Leu Glu Trp Glu Pro Lys Pro Val Asn Gln 20 25 30 Val Tyr Thr
Val Gln Ile Ser Thr Lys Ser Gly Asp Trp Lys Ser Lys 35 40 45 Cys
Phe Tyr Thr Thr Asp Thr Glu Cys Asp Leu Thr Asp Glu Ile Val 50 55
60 Lys Asp Val Lys Gln Thr Tyr Leu Ala Arg Val Phe Ser Tyr Pro Ala
65 70 75 80 Gly Asn Val Glu Ser Thr Gly Ser Ala Gly Glu Pro Leu Tyr
Glu Asn 85 90 95 Ser Pro Glu Phe Thr Pro Tyr Leu Glu Thr Asn Leu
Gly Gln Pro Thr 100 105 110 Ile Gln Ser Phe Glu Gln Val Gly Thr Lys
Val Asn Val Thr Val Glu 115 120 125 Asp Glu Arg Thr Leu Val Arg Arg
Asn Asn Thr Phe Leu Ser Leu Arg 130 135 140 Asp Val Phe Gly Lys Asp
Leu Ile Tyr Thr Leu Tyr Tyr Trp Lys Ser 145 150 155 160 Ser Ser Ser
Gly Lys Lys Thr Ala Lys Thr Asn Thr Asn Glu Phe Leu 165 170 175 Ile
Asp Val Asp Lys Gly Glu Asn Tyr Cys Phe Ser Val Gln Ala Val 180 185
190 Ile Pro Ser Arg Thr Val Asn Arg Lys Ser Thr Asp Ser Pro Val Glu
195 200 205 Cys Met Gly Gln Glu Lys Gly Glu Phe Arg Glu Ile Phe Tyr
Ile Ile 210 215 220 Gly Ala Val Val Phe Val Val Ile Ile Leu Val Ile
Ile Leu Ala Ile 225 230 235 240 Ser Leu His Lys Cys Arg Lys Ala Gly
Val Gly Gln Ser Trp Lys Glu 245 250 255 Asn Ser Pro Leu Asn Val Ser
260 3 219 PRT Homo sapiens 3 Ser Gly Thr Thr Asn Thr Val Ala Ala
Tyr Asn Leu Thr Trp Lys Ser 1 5 10 15 Thr Asn Phe Lys Thr Ile Leu
Glu Trp Glu Pro Lys Pro Val Asn Gln 20 25 30 Val Tyr Thr Val Gln
Ile Ser Thr Lys Ser Gly Asp Trp Lys Ser Lys 35 40 45 Cys Phe Tyr
Thr Thr Asp Thr Glu Cys Asp Leu Thr Asp Glu Ile Val 50 55 60 Lys
Asp Val Lys Gln Thr Tyr Leu Ala Arg Val Phe Ser Tyr Pro Ala 65 70
75 80 Gly Asn Val Glu Ser Thr Gly Ser Ala Gly Glu Pro Leu Tyr Glu
Asn 85 90 95 Ser Pro Glu Phe Thr Pro Tyr Leu Glu Thr Asn Leu Gly
Gln Pro Thr 100 105 110 Ile Gln Ser Phe Glu Gln Val Gly Thr Lys Val
Asn Val Thr Val Glu 115 120 125 Asp Glu Arg Thr Leu Val Arg Arg Asn
Asn Thr Phe Leu Ser Leu Arg 130 135 140 Asp Val Phe Gly Lys Asp Leu
Ile Tyr Thr Leu Tyr Tyr Trp Lys Ser 145 150 155 160 Ser Ser Ser Gly
Lys Lys Thr Ala Lys Thr Asn Thr Asn Glu Phe Leu 165 170 175 Ile Asp
Val Asp Lys Gly Glu Asn Tyr Cys Phe Ser Val Gln Ala Val 180 185 190
Ile Pro Ser Arg Thr Val Asn Arg Lys Ser Thr Asp Ser Pro Val Glu 195
200 205 Cys Met Gly Gln Glu Lys Gly Glu Phe Arg Glu 210 215 4 218
PRT Homo sapiens 4 Ser Gly Thr Thr Asn Thr Val Ala Ala Tyr Asn Leu
Thr Trp Lys Ser 1 5 10 15 Thr Asn Phe Lys Thr Ile Leu Glu Trp Glu
Pro Lys Pro Val Asn Gln 20 25 30 Val Tyr Thr Val Gln Ile Ser Thr
Lys Ser Gly Asp Trp Lys Ser Lys 35 40 45 Cys Phe Tyr Thr Thr Asp
Thr Glu Cys Asp Leu Thr Asp Glu Ile Val 50 55 60 Lys Asp Val Lys
Gln Thr Tyr Leu Ala Arg Val Phe Ser Tyr Pro Ala 65 70 75 80 Gly Asn
Val Glu Ser Thr Gly Ser Ala Gly Glu Pro Leu Tyr Glu Asn 85 90 95
Ser Pro Glu Phe Thr Pro Tyr Leu Glu Thr Asn Leu Gly Gln Pro Thr 100
105 110 Ile Gln Ser Phe Glu Gln Val Gly Thr Lys Val Asn Val Thr Val
Glu 115 120 125 Asp Glu Arg Thr Leu Val Arg Arg Asn Asn Thr Phe Leu
Ser Leu Arg 130 135 140 Asp Val Phe Gly Lys Asp Leu Ile Tyr Thr Leu
Tyr Tyr Trp Lys Ser 145 150 155 160 Ser Ser Ser Gly Lys Lys Thr Ala
Lys Thr Asn Thr Asn Glu Phe Leu 165 170 175 Ile Asp Val Asp Lys Gly
Glu Asn Tyr Cys Phe Ser Val Gln Ala Val 180 185 190 Ile Pro Ser Arg
Thr Val Asn Arg Lys Ser Thr Asp Ser Pro Val Glu 195 200 205 Cys Met
Gly Gln Glu Lys Gly Glu Phe Arg 210 215 5 217 PRT Homo sapiens 5
Thr Thr Asn Thr Val Ala Ala Tyr Asn Leu Thr Trp Lys Ser Thr Asn 1 5
10 15 Phe Lys Thr Ile Leu Glu Trp Glu Pro Lys Pro Val Asn Gln Val
Tyr 20 25 30 Thr Val Gln Ile Ser Thr Lys Ser Gly Asp Trp Lys Ser
Lys Cys Phe 35 40 45 Tyr Thr Thr Asp Thr Glu Cys Asp Leu Thr Asp
Glu Ile Val Lys Asp 50 55 60 Val Lys Gln Thr Tyr Leu Ala Arg Val
Phe Ser Tyr Pro Ala Gly Asn 65 70 75 80 Val Glu Ser Thr Gly Ser Ala
Gly Glu Pro Leu Tyr Glu Asn Ser Pro 85 90 95 Glu Phe Thr Pro Tyr
Leu Glu Thr Asn Leu Gly Gln Pro Thr Ile Gln 100 105 110 Ser Phe Glu
Gln Val Gly Thr Lys Val Asn Val Thr Val Glu Asp Glu 115 120 125 Arg
Thr Leu Val Arg Arg Asn Asn Thr Phe Leu Ser Leu Arg Asp Val 130 135
140 Phe Gly Lys Asp Leu Ile Tyr Thr Leu Tyr Tyr Trp Lys Ser Ser Ser
145 150 155 160 Ser Gly Lys Lys Thr Ala Lys Thr Asn Thr Asn Glu Phe
Leu Ile Asp 165 170 175 Val Asp Lys Gly Glu Asn Tyr Cys Phe Ser Val
Gln Ala Val Ile Pro 180 185 190 Ser Arg Thr Val Asn Arg Lys Ser Thr
Asp Ser Pro Val Glu Cys Met 195 200 205 Gly Gln Glu Lys Gly Glu Phe
Arg Glu 210 215 6 216 PRT Homo sapiens 6 Thr Thr Asn Thr Val Ala
Ala Tyr Asn Leu Thr Trp Lys Ser Thr Asn 1 5 10 15 Phe Lys Thr Ile
Leu Glu Trp Glu Pro Lys Pro Val Asn Gln Val Tyr 20 25 30 Thr Val
Gln Ile Ser Thr Lys Ser Gly Asp Trp Lys Ser Lys Cys Phe 35 40 45
Tyr Thr Thr Asp Thr Glu Cys Asp Leu Thr Asp Glu Ile Val Lys Asp 50
55 60 Val Lys Gln Thr Tyr Leu Ala Arg Val Phe Ser Tyr Pro Ala Gly
Asn 65 70 75 80 Val Glu Ser Thr Gly Ser Ala Gly Glu Pro Leu Tyr Glu
Asn Ser Pro 85 90 95 Glu Phe Thr Pro Tyr Leu Glu Thr Asn Leu Gly
Gln Pro Thr Ile Gln 100 105 110 Ser Phe Glu Gln Val Gly Thr Lys Val
Asn Val Thr Val Glu Asp Glu 115 120 125 Arg Thr Leu Val Arg Arg Asn
Asn Thr Phe Leu Ser Leu Arg Asp Val 130 135 140 Phe Gly Lys Asp Leu
Ile Tyr Thr Leu Tyr Tyr Trp Lys Ser Ser Ser 145 150 155 160 Ser Gly
Lys Lys Thr Ala Lys Thr Asn Thr Asn Glu Phe Leu Ile Asp 165 170 175
Val Asp Lys Gly Glu Asn Tyr Cys Phe Ser Val Gln Ala Val Ile Pro 180
185 190 Ser Arg Thr Val Asn Arg Lys Ser Thr Asp Ser Pro Val Glu Cys
Met 195 200 205 Gly Gln Glu Lys Gly Glu Phe Arg 210 215 7 22 PRT
Homo sapiens 7 Phe Tyr Ile Ile Gly Ala Val Val Phe Val Val Ile Ile
Leu Val Ile 1 5 10 15 Ile Leu Ala Ile Ser Leu 20 8 2320 PRT Homo
sapiens 8 Met Val Gln Pro Gln Ser Pro Val Ala Val Ser Gln Ser Lys
Pro Gly 1 5 10 15 Cys Tyr Asp Asn Gly Lys His Tyr Gln Ile Asn Gln
Gln Trp Glu Arg 20 25 30 Thr Tyr Leu Gly Asn Ala Leu Val Cys Thr
Cys Tyr Gly Gly Ser Arg 35 40 45 Gly Phe Asn Cys Glu Ser Lys Pro
Glu Ala Glu Glu Thr Cys Phe Asp 50 55 60 Lys Tyr Thr Gly Asn Thr
Tyr Arg Val Gly Asp Thr Tyr Glu Arg Pro 65 70 75 80 Lys Asp Ser Met
Ile Trp Asp Cys Thr Cys Ile Gly Ala Gly Arg Gly 85 90 95 Arg Ile
Ser Cys Thr Ile Ala Asn Arg Cys His Glu Gly Gly Gln Ser 100 105 110
Tyr Lys Ile Gly Asp Thr Trp Arg Arg Pro His Glu Thr Gly Gly Tyr 115
120 125 Met Leu Glu Cys Val Cys Leu Gly Asn Gly Lys Gly Glu Trp Thr
Cys 130 135 140 Lys Pro Ile Ala Glu Lys Cys Phe Asp His Ala Ala Gly
Thr Ser Tyr 145 150 155 160 Val Val Gly Glu Thr Trp Glu Lys Pro Tyr
Gln Gly Trp Met Met Val 165 170 175 Asp Cys Thr Cys Leu Gly Glu Gly
Ser Gly Arg Ile Thr Cys Thr Ser 180 185 190 Arg Asn Arg Cys Asn Asp
Gln Asp Thr Arg Thr Ser Tyr Arg Ile Gly 195 200 205 Asp Thr Trp Ser
Lys Lys Asp Asn Arg Gly Asn Leu Leu Gln Cys Ile 210 215 220 Cys Thr
Gly Asn Gly Arg Gly Glu Trp Lys Cys Glu Arg His Thr Ser 225 230 235
240 Val Gln Thr Thr Ser Ser Gly Ser Gly Pro Phe Thr Asp Val Arg Ala
245 250 255 Ala Val Tyr Gln Pro Gln Pro His Pro Gln Pro Pro Pro Tyr
Gly His 260 265 270 Cys Val Thr Asp Ser Gly Val Val Tyr Ser Val Gly
Met Gln Trp Leu 275 280 285 Lys Thr Gln Gly Asn Lys Gln Met Leu Cys
Thr Cys Leu Gly Asn Gly 290 295 300 Val Ser Cys Gln Glu Thr Ala Val
Thr Gln Thr Tyr Gly Gly Asn Ser 305 310 315 320 Asn Gly Glu Pro Cys
Val Leu Pro Phe Thr Tyr Asn Gly Arg Thr Phe 325 330 335 Tyr Ser Cys
Thr Thr Glu Gly Arg Gln Asp Gly His Leu Trp Cys Ser 340 345 350 Thr
Thr Ser Asn Tyr Glu Gln Asp Gln Lys Tyr Ser Phe Cys Thr Asp 355 360
365 His Thr Val Leu Val Gln Thr Arg Gly Gly Asn Ser Asn Gly Ala Leu
370 375 380 Cys His Phe Pro Phe Leu Tyr Asn Asn His Asn Tyr Thr Asp
Cys Thr 385 390 395 400 Ser Glu Gly Arg Arg Asp Asn Met Lys Trp Cys
Gly Thr Thr Gln Asn 405 410 415 Tyr Asp Ala Asp Gln Lys Phe Gly Phe
Cys Pro Met Ala Ala His Glu 420 425 430 Glu Ile Cys Thr Thr Asn Glu
Gly Val Met Tyr Arg Ile Gly Asp Gln 435 440 445 Trp Asp Lys Gln His
Asp Met Gly His Met Met Arg Cys Thr Cys Val 450 455 460 Gly Asn Gly
Arg Gly Glu Trp Thr Cys Ile Ala Tyr Ser Gln Leu Arg 465 470 475 480
Asp Gln Cys Ile Val Asp Asp Ile Thr Tyr Asn Val Asn Asp Thr Phe 485
490 495 His Lys Arg His Glu Glu Gly His Met Leu Asn Cys Thr Cys Phe
Gly 500 505 510 Gln Gly Arg Gly Arg Trp Lys Cys Asp Pro Val Asp Gln
Cys Gln Asp 515 520 525 Ser Glu Thr Gly Thr Phe Tyr Gln Ile Gly Asp
Ser Trp Glu Lys Tyr 530 535 540 Val His Gly Val Arg Tyr Gln Cys Tyr
Cys Tyr Gly Arg Gly Ile Gly 545 550 555 560 Glu Trp His Cys Gln Pro
Leu Gln Thr Tyr Pro Ser Ser Ser Gly Pro 565 570 575 Val Glu Val Phe
Ile Thr Glu Thr Pro Ser Gln Pro Asn Ser His Pro 580 585 590 Ile Gln
Trp Asn Ala Pro Gln Pro Ser His Ile Ser Lys Tyr Ile Leu 595 600 605
Arg Trp Arg Pro Lys Asn Ser Val Gly Arg Trp Lys Glu Ala Thr Ile 610
615 620 Pro Gly His Leu Asn Ser Tyr Thr Ile Lys Gly Leu Lys Pro Gly
Val 625 630 635 640 Val Tyr Glu Gly Gln Leu Ile Ser Ile Gln Gln Tyr
Gly His Gln Glu 645 650 655 Val Thr Arg Phe Asp Phe Thr Thr Thr Ser
Thr Ser Thr Pro Val Thr 660 665 670 Ser Asn Thr Val Thr Gly Glu Thr
Thr Pro Phe Ser Pro Leu Val Ala 675 680 685 Thr Ser Glu Ser Val Thr
Glu Ile Thr Ala Ser Ser Phe Val Val Ser 690 695 700 Trp Val Ser Ala
Ser Asp Thr Val Ser Gly Phe Arg Val Glu Tyr Glu 705 710 715 720 Leu
Ser Glu Glu Gly Asp Glu Pro Gln Tyr Leu Asp Leu Pro Ser Thr 725 730
735 Ala Thr Ser Val Asn Ile Pro Asp Leu Leu Pro Gly Arg Lys Tyr Ile
740 745 750 Val Asn Val Tyr Gln Ile Ser Glu Asp Gly Glu Gln Ser Leu
Ile Leu 755 760 765 Ser Thr Ser Gln Thr Thr Ala Pro Asp Ala Pro Pro
Asp Thr Thr Val 770 775 780 Asp Gln Val Asp Asp Thr Ser Ile Val Val
Arg Trp Ser Arg Pro Gln 785 790 795 800 Ala Pro Ile Thr Gly Tyr Arg
Ile Val Tyr Ser Pro Ser Val Glu Gly 805 810 815 Ser Ser Thr Glu Leu
Asn Leu Pro Glu Thr Ala Asn Ser Val Thr Leu 820 825 830 Ser Asp Leu
Gln Pro Gly Val Gln Tyr Asn Ile Thr Ile Tyr Ala Val 835 840 845 Glu
Glu Asn Gln Glu Ser Thr Pro Val Val Ile Gln Gln Glu Thr Thr 850 855
860 Gly Thr Pro Arg Ser Asp Thr Val Pro Ser Pro Arg Asp Leu Gln Phe
865 870 875 880 Val Glu Val Thr Asp Val Lys Val Thr Ile Met Trp Thr
Pro Pro Glu 885 890 895 Ser Ala Val Thr Gly Tyr Arg Val Asp Val Ile
Pro Val Asn Leu Pro 900 905 910 Gly Glu His Gly Gln Arg Leu Pro Ile
Ser Arg Asn Thr Phe Ala Glu 915 920 925 Val Thr Gly Leu Ser Pro Gly
Val Thr Tyr Tyr Phe Lys Val Phe Ala 930 935 940 Val Ser His Gly Arg
Glu Ser Lys Pro Leu Thr Ala Gln Gln Thr Thr 945 950 955 960 Lys Leu
Asp Ala Pro Thr Asn Leu Gln Phe Val Asn Glu Thr Asp Ser 965 970 975
Thr Val Leu Val
Arg Trp Thr Pro Pro Arg Ala Gln Ile Thr Gly Tyr 980 985 990 Arg Leu
Thr Val Gly Leu Thr Arg Arg Gly Gln Pro Arg Gln Tyr Asn 995 1000
1005 Val Gly Pro Ser Val Ser Lys Tyr Pro Leu Arg Asn Leu Gln Pro
Ala 1010 1015 1020 Ser Glu Tyr Thr Val Ser Leu Val Ala Ile Lys Gly
Asn Gln Glu Ser 1025 1030 1035 1040 Pro Lys Ala Thr Gly Val Phe Thr
Thr Leu Gln Pro Gly Ser Ser Ile 1045 1050 1055 Pro Pro Tyr Asn Thr
Glu Val Thr Glu Thr Thr Ile Val Ile Thr Trp 1060 1065 1070 Thr Pro
Ala Pro Arg Ile Gly Phe Lys Leu Gly Val Arg Pro Ser Gln 1075 1080
1085 Gly Gly Glu Ala Pro Arg Glu Val Thr Ser Asp Ser Gly Ser Ile
Val 1090 1095 1100 Val Ser Gly Leu Thr Pro Gly Val Glu Tyr Val Tyr
Thr Ile Gln Val 1105 1110 1115 1120 Leu Arg Asp Gly Gln Glu Arg Asp
Ala Pro Ile Val Asn Lys Val Val 1125 1130 1135 Thr Pro Leu Ser Pro
Pro Thr Asn Leu His Leu Glu Ala Asn Pro Asp 1140 1145 1150 Thr Gly
Val Leu Thr Val Ser Trp Glu Arg Ser Thr Thr Pro Asp Ile 1155 1160
1165 Thr Gly Tyr Arg Ile Thr Thr Thr Pro Thr Asn Gly Gln Gln Gly
Asn 1170 1175 1180 Ser Leu Glu Glu Val Val His Ala Asp Gln Ser Ser
Cys Thr Phe Asp 1185 1190 1195 1200 Asn Leu Ser Pro Gly Leu Glu Tyr
Asn Val Ser Val Tyr Thr Val Lys 1205 1210 1215 Asp Asp Lys Glu Ser
Val Pro Ile Ser Asp Thr Ile Ile Pro Ala Val 1220 1225 1230 Pro Pro
Pro Thr Asp Leu Arg Phe Thr Asn Ile Gly Pro Asp Thr Met 1235 1240
1245 Arg Val Thr Trp Ala Pro Pro Pro Ser Ile Asp Leu Thr Asn Phe
Leu 1250 1255 1260 Val Arg Tyr Ser Pro Val Lys Asn Glu Glu Asp Val
Ala Glu Leu Ser 1265 1270 1275 1280 Ile Ser Pro Ser Asp Asn Ala Val
Val Leu Thr Asn Leu Leu Pro Gly 1285 1290 1295 Thr Glu Tyr Val Val
Ser Val Ser Ser Val Tyr Glu Gln His Glu Ser 1300 1305 1310 Thr Pro
Leu Arg Gly Arg Gln Lys Thr Gly Leu Asp Ser Pro Thr Gly 1315 1320
1325 Ile Asp Phe Ser Asp Ile Thr Ala Asn Ser Phe Thr Val His Trp
Ile 1330 1335 1340 Ala Pro Arg Ala Thr Ile Thr Gly Tyr Arg Ile Arg
His His Pro Glu 1345 1350 1355 1360 His Phe Ser Gly Arg Pro Arg Glu
Asp Arg Val Pro His Ser Arg Asn 1365 1370 1375 Ser Ile Thr Leu Thr
Asn Leu Thr Pro Gly Thr Glu Tyr Val Val Ser 1380 1385 1390 Ile Val
Ala Leu Asn Gly Arg Glu Glu Ser Pro Leu Leu Ile Gly Gln 1395 1400
1405 Gln Ser Thr Val Ser Asp Val Pro Arg Asp Leu Glu Val Val Ala
Ala 1410 1415 1420 Thr Pro Thr Ser Leu Leu Ile Ser Trp Asp Ala Pro
Ala Val Thr Val 1425 1430 1435 1440 Arg Tyr Tyr Arg Ile Thr Tyr Gly
Glu Thr Gly Gly Asn Ser Pro Val 1445 1450 1455 Gln Glu Phe Thr Val
Pro Gly Ser Lys Ser Thr Ala Thr Ile Ser Gly 1460 1465 1470 Leu Lys
Pro Gly Val Asp Tyr Thr Ile Thr Val Tyr Ala Val Thr Gly 1475 1480
1485 Arg Gly Asp Ser Pro Ala Ser Ser Lys Pro Ile Ser Ile Asn Tyr
Arg 1490 1495 1500 Thr Glu Ile Asp Lys Pro Ser Gln Met Gln Val Thr
Asp Val Gln Asp 1505 1510 1515 1520 Asn Ser Ile Ser Val Lys Trp Leu
Pro Ser Ser Ser Pro Val Thr Gly 1525 1530 1535 Tyr Arg Val Thr Thr
Thr Pro Lys Asn Gly Pro Gly Pro Thr Lys Thr 1540 1545 1550 Lys Thr
Ala Gly Pro Asp Gln Thr Glu Met Thr Ile Glu Gly Leu Gln 1555 1560
1565 Pro Thr Val Glu Tyr Val Val Ser Val Tyr Ala Gln Asn Pro Ser
Gly 1570 1575 1580 Glu Ser Gln Pro Leu Val Gln Thr Ala Val Thr Asn
Ile Asp Arg Pro 1585 1590 1595 1600 Lys Gly Leu Ala Phe Thr Asp Val
Asp Val Asp Ser Ile Lys Ile Ala 1605 1610 1615 Trp Glu Ser Pro Gln
Gly Gln Val Ser Arg Tyr Arg Val Thr Tyr Ser 1620 1625 1630 Ser Pro
Glu Asp Gly Ile His Glu Leu Phe Pro Ala Pro Asp Gly Glu 1635 1640
1645 Glu Asp Thr Ala Glu Leu Gln Gly Leu Arg Pro Gly Ser Glu Tyr
Thr 1650 1655 1660 Val Ser Val Val Ala Leu His Asp Asp Met Glu Ser
Gln Pro Leu Ile 1665 1670 1675 1680 Gly Thr Gln Ser Thr Ala Ile Pro
Ala Pro Thr Asp Leu Lys Phe Thr 1685 1690 1695 Gln Val Thr Pro Thr
Ser Leu Ser Ala Gln Trp Thr Pro Pro Asn Val 1700 1705 1710 Gln Leu
Thr Gly Tyr Arg Val Arg Val Thr Pro Lys Glu Lys Thr Gly 1715 1720
1725 Pro Met Lys Glu Ile Asn Leu Ala Pro Asp Ser Ser Ser Val Val
Val 1730 1735 1740 Ser Gly Leu Met Val Ala Thr Lys Tyr Glu Val Ser
Val Tyr Ala Leu 1745 1750 1755 1760 Lys Asp Thr Leu Thr Ser Arg Pro
Ala Gln Gly Val Val Thr Thr Leu 1765 1770 1775 Glu Asn Val Ser Pro
Pro Arg Arg Ala Arg Val Thr Asp Ala Thr Glu 1780 1785 1790 Thr Thr
Ile Thr Ile Ser Trp Arg Thr Lys Thr Glu Thr Ile Thr Gly 1795 1800
1805 Phe Gln Val Asp Ala Val Pro Ala Asn Gly Gln Thr Pro Ile Gln
Arg 1810 1815 1820 Thr Ile Lys Pro Asp Val Arg Ser Tyr Thr Ile Thr
Gly Leu Gln Pro 1825 1830 1835 1840 Gly Thr Asp Tyr Lys Ile Tyr Leu
Tyr Thr Leu Asn Asp Asn Ala Arg 1845 1850 1855 Ser Ser Pro Val Val
Ile Asp Ala Ser Thr Ala Ile Asp Ala Pro Ser 1860 1865 1870 Asn Leu
Arg Phe Leu Ala Thr Thr Pro Asn Ser Leu Leu Val Ser Trp 1875 1880
1885 Gln Pro Pro Arg Ala Arg Ile Thr Gly Tyr Ile Ile Lys Tyr Glu
Lys 1890 1895 1900 Pro Gly Ser Pro Pro Arg Glu Val Val Pro Arg Pro
Arg Pro Gly Val 1905 1910 1915 1920 Thr Glu Ala Thr Ile Thr Gly Leu
Glu Pro Gly Thr Glu Tyr Thr Ile 1925 1930 1935 Tyr Val Ile Ala Leu
Lys Asn Asn Gln Lys Ser Glu Pro Leu Ile Gly 1940 1945 1950 Arg Lys
Lys Thr Asp Glu Leu Pro Gln Leu Val Thr Leu Pro His Pro 1955 1960
1965 Asn Leu His Gly Pro Glu Ile Leu Asp Val Pro Ser Thr Val Gln
Lys 1970 1975 1980 Thr Pro Phe Val Thr His Pro Gly Tyr Asp Thr Gly
Asn Gly Ile Gln 1985 1990 1995 2000 Leu Pro Gly Thr Ser Gly Gln Gln
Pro Ser Val Gly Gln Gln Met Ile 2005 2010 2015 Phe Glu Glu His Gly
Phe Arg Arg Thr Thr Pro Pro Thr Thr Ala Thr 2020 2025 2030 Pro Ile
Arg His Arg Pro Arg Pro Tyr Pro Pro Asn Val Gly Gln Glu 2035 2040
2045 Ala Leu Ser Gln Thr Thr Ile Ser Trp Ala Pro Phe Gln Asp Thr
Ser 2050 2055 2060 Glu Tyr Ile Ile Ser Cys His Pro Val Gly Thr Asp
Glu Glu Pro Leu 2065 2070 2075 2080 Gln Phe Arg Val Pro Gly Thr Ser
Thr Ser Ala Thr Leu Thr Gly Leu 2085 2090 2095 Thr Arg Gly Ala Thr
Tyr Asn Val Ile Val Glu Ala Leu Lys Asp Gln 2100 2105 2110 Gln Arg
His Lys Val Arg Glu Glu Val Val Thr Val Gly Asn Ser Val 2115 2120
2125 Asn Glu Gly Leu Asn Gln Pro Thr Asp Asp Ser Cys Phe Asp Pro
Tyr 2130 2135 2140 Thr Val Ser His Tyr Ala Val Gly Asp Glu Trp Glu
Arg Met Ser Glu 2145 2150 2155 2160 Ser Gly Phe Lys Leu Leu Cys Gln
Cys Leu Gly Phe Gly Ser Gly His 2165 2170 2175 Phe Arg Cys Asp Ser
Ser Arg Trp Cys His Asp Asn Gly Val Asn Tyr 2180 2185 2190 Lys Ile
Gly Glu Lys Trp Asp Arg Gln Gly Glu Asn Gly Gln Met Met 2195 2200
2205 Ser Cys Thr Cys Leu Gly Asn Gly Lys Gly Glu Phe Lys Cys Asp
Pro 2210 2215 2220 His Glu Ala Thr Cys Tyr Asp Asp Gly Lys Thr Tyr
His Val Gly Glu 2225 2230 2235 2240 Gln Trp Gln Lys Glu Tyr Leu Gly
Ala Ile Cys Ser Cys Thr Cys Phe 2245 2250 2255 Gly Gly Gln Arg Gly
Trp Arg Cys Asp Asn Cys Arg Arg Pro Gly Gly 2260 2265 2270 Glu Pro
Ser Pro Glu Gly Thr Thr Gly Gln Ser Tyr Asn Gln Tyr Ser 2275 2280
2285 Gln Arg Tyr His Gln Arg Thr Asn Thr Asn Val Asn Cys Pro Ile
Glu 2290 2295 2300 Cys Phe Met Pro Leu Asp Val Gln Ala Asp Arg Glu
Asp Ser Arg Glu 2305 2310 2315 2320 9 599 PRT Homo sapiens 9 Met
Arg Gly Ser His His His His His His Gly Ser Gly Ser Ser Thr 1 5 10
15 Pro Pro Pro Thr Asp Leu Arg Phe Thr Asn Ile Gly Pro Asp Thr Met
20 25 30 Arg Val Thr Trp Ala Pro Pro Pro Ser Ile Asp Leu Thr Asn
Phe Leu 35 40 45 Val Arg Tyr Ser Pro Val Lys Asn Glu Glu Asp Val
Ala Glu Leu Ser 50 55 60 Ile Ser Pro Ser Asp Asn Ala Val Val Leu
Thr Asn Leu Leu Pro Gly 65 70 75 80 Thr Glu Tyr Val Val Ser Val Ser
Ser Val Tyr Glu Gln His Glu Ser 85 90 95 Thr Pro Leu Arg Gly Arg
Gln Lys Thr Gly Leu Asp Ser Pro Thr Gly 100 105 110 Ile Asp Phe Ser
Asp Ile Thr Ala Asn Ser Phe Thr Val His Trp Ile 115 120 125 Ala Pro
Arg Ala Thr Ile Thr Gly Tyr Arg Ile Arg His His Pro Glu 130 135 140
His Phe Ser Gly Arg Pro Arg Glu Asp Arg Val Pro His Ser Arg Asn 145
150 155 160 Ser Ile Thr Leu Thr Asn Leu Thr Pro Gly Thr Glu Tyr Val
Val Ser 165 170 175 Ile Val Ala Leu Asn Gly Arg Glu Glu Ser Pro Leu
Leu Ile Gly Gln 180 185 190 Gln Ser Thr Val Ser Asp Val Pro Arg Asp
Leu Glu Val Val Ala Ala 195 200 205 Thr Pro Thr Ser Leu Leu Ile Ser
Trp Asp Ala Pro Ala Val Thr Val 210 215 220 Arg Tyr Tyr Arg Ile Thr
Tyr Gly Glu Thr Gly Gly Asn Ser Pro Val 225 230 235 240 Gln Glu Phe
Thr Val Pro Gly Ser Lys Ser Thr Ala Thr Ile Ser Gly 245 250 255 Leu
Lys Pro Gly Val Asp Tyr Thr Ile Thr Val Tyr Ala Val Thr Gly 260 265
270 Arg Gly Asp Ser Pro Ala Ser Ser Lys Pro Ile Ser Ile Asn Tyr Arg
275 280 285 Thr Glu Ile Asp Lys Pro Ser Gln Met Gln Val Thr Asp Val
Gln Asp 290 295 300 Asn Ser Ile Ser Val Lys Trp Leu Pro Ser Ser Ser
Pro Val Thr Gly 305 310 315 320 Tyr Arg Val Thr Thr Thr Pro Lys Asn
Gly Pro Gly Pro Thr Lys Thr 325 330 335 Lys Thr Ala Gly Pro Asp Gln
Thr Glu Met Thr Ile Glu Gly Leu Gln 340 345 350 Pro Thr Val Glu Tyr
Val Val Ser Val Tyr Ala Gln Asn Pro Ser Gly 355 360 365 Glu Ser Gln
Pro Leu Val Gln Thr Ala Val Thr Ser Ser Ser Gly Thr 370 375 380 Thr
Asn Thr Val Ala Ala Tyr Asn Leu Thr Trp Lys Ser Thr Asn Phe 385 390
395 400 Lys Thr Ile Leu Glu Trp Glu Pro Lys Pro Val Asn Gln Val Tyr
Thr 405 410 415 Val Gln Ile Ser Thr Lys Ser Gly Asp Trp Lys Ser Lys
Cys Phe Tyr 420 425 430 Thr Thr Asp Thr Glu Cys Asp Leu Thr Asp Glu
Ile Val Lys Asp Val 435 440 445 Lys Gln Thr Tyr Leu Ala Arg Val Phe
Ser Tyr Pro Ala Gly Asn Val 450 455 460 Glu Ser Thr Gly Ser Ala Gly
Glu Pro Leu Tyr Glu Asn Ser Pro Glu 465 470 475 480 Phe Thr Pro Tyr
Leu Glu Thr Asn Leu Gly Gln Pro Thr Ile Gln Ser 485 490 495 Phe Glu
Gln Val Gly Thr Lys Val Asn Val Thr Val Glu Asp Glu Arg 500 505 510
Thr Leu Val Arg Arg Asn Asn Thr Phe Leu Ser Leu Arg Asp Val Phe 515
520 525 Gly Lys Asp Leu Ile Tyr Thr Leu Tyr Tyr Trp Lys Ser Ser Ser
Ser 530 535 540 Gly Lys Lys Thr Ala Lys Thr Asn Thr Asn Glu Phe Leu
Ile Asp Val 545 550 555 560 Asp Lys Gly Glu Asn Tyr Cys Phe Ser Val
Gln Ala Val Ile Pro Ser 565 570 575 Arg Thr Val Asn Arg Lys Ser Thr
Asp Ser Pro Val Glu Cys Met Gly 580 585 590 Gln Glu Lys Gly Glu Phe
Arg 595 10 330 PRT Homo sapiens 10 Met Arg Gly Ser His His His His
His His Gly Ser Gly Ser Ser Thr 1 5 10 15 Val Ser Asp Val Pro Arg
Asp Leu Glu Val Val Ala Ala Thr Pro Thr 20 25 30 Ser Leu Leu Ile
Ser Trp Asp Ala Pro Ala Val Thr Val Arg Tyr Tyr 35 40 45 Arg Ile
Thr Tyr Gly Glu Thr Gly Gly Asn Ser Pro Val Gln Glu Phe 50 55 60
Thr Val Pro Gly Ser Lys Ser Thr Ala Thr Ile Ser Gly Leu Lys Pro 65
70 75 80 Gly Val Asp Tyr Thr Ile Thr Val Tyr Ala Val Thr Gly Arg
Gly Asp 85 90 95 Ser Pro Ala Ser Ser Lys Pro Ile Ser Ile Asn Tyr
Arg Thr Ser Ser 100 105 110 Ser Gly Thr Thr Asn Thr Val Ala Ala Tyr
Asn Leu Thr Trp Lys Ser 115 120 125 Thr Asn Phe Lys Thr Ile Leu Glu
Trp Glu Pro Lys Pro Val Asn Gln 130 135 140 Val Tyr Thr Val Gln Ile
Ser Thr Lys Ser Gly Asp Trp Lys Ser Lys 145 150 155 160 Cys Phe Tyr
Thr Thr Asp Thr Glu Cys Asp Leu Thr Asp Glu Ile Val 165 170 175 Lys
Asp Val Lys Gln Thr Tyr Leu Ala Arg Val Phe Ser Tyr Pro Ala 180 185
190 Gly Asn Val Glu Ser Thr Gly Ser Ala Gly Glu Pro Leu Tyr Glu Asn
195 200 205 Ser Pro Glu Phe Thr Pro Tyr Leu Glu Thr Asn Leu Gly Gln
Pro Thr 210 215 220 Ile Gln Ser Phe Glu Gln Val Gly Thr Lys Val Asn
Val Thr Val Glu 225 230 235 240 Asp Glu Arg Thr Leu Val Arg Arg Asn
Asn Thr Phe Leu Ser Leu Arg 245 250 255 Asp Val Phe Gly Lys Asp Leu
Ile Tyr Thr Leu Tyr Tyr Trp Lys Ser 260 265 270 Ser Ser Ser Gly Lys
Lys Thr Ala Lys Thr Asn Thr Asn Glu Phe Leu 275 280 285 Ile Asp Val
Asp Lys Gly Glu Asn Tyr Cys Phe Ser Val Gln Ala Val 290 295 300 Ile
Pro Ser Arg Thr Val Asn Arg Lys Ser Thr Asp Ser Pro Val Glu 305 310
315 320 Cys Met Gly Gln Glu Lys Gly Glu Phe Arg 325 330 11 24 DNA
Artificial Sequence A primer. 11 caccaacaac ttgcatctgg aggc 24 12
24 DNA Artificial Sequence A primer. 12 aacattgggt ggtgtccact gggc
24 13 48 DNA Artificial Sequence A primer. 13 accatcacgg atccggggtc
gtcgacacct cctcccactg acctgcga 48 14 40 DNA Artificial Sequence A
primer. 14 ggtaccggag gagctcgtta cctgcagtct gaaccagagg 40 15 36 DNA
Artificial Sequence A primer. 15 acgagctcct ccggtaccac aaatactgtg
ggcagc 36 16 20 DNA Artificial Sequence A primer. 16 tctgcgttct
gatttaatct 20 17 4 PRT Artificial Sequence A RGDS peptide. 17 Arg
Gly Asp Ser 1 18 19 PRT Artificial Sequence A peptide containing a
poly His tag and a processing protease (Fxa) cleavage site followed
by a cysteine. 18 Met Xaa Xaa Xaa His His His His His His Xaa Xaa
Xaa Xaa Ile Glu 1 5 10 15 Gly Arg Cys 19 5 PRT Artificial Sequence
A lysine containing linker. 19 Lys Ser Gly Gly Gly
1 5 20 21 DNA Artificial Sequence A primer. 20 actacaaata
ctgtggcagc a 21 21 33 DNA Artificial Sequence A primer. 21
tttaagcttt cacgtgccca tacactctac cgg 33 22 50 DNA Artificial
Sequence A primer. 22 aaatggatcc tggtgcctag gggcccggga ctacaaatac
tgtggcagca 50 23 5 PRT Artificial Sequence A thrombin cleavage
site. 23 Val Pro Arg Gly Ser 1 5 24 15 PRT Artificial Sequence A
linker sequence with three repeats of Gly4Ser. 24 Gly Gly Gly Gly
Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 1 5 10 15 25 37 DNA
Artificial Sequence A primer. 25 gatcttggtc cctaggggat ccgcagaacc
aatgcct 37 26 36 DNA Artificial Sequence A primer. 26 cactcgctaa
acttcagtca atacctctgg tatact 36 27 36 DNA Artificial Sequence A
primer. 27 ggtaccggag gaggcggttc aggtggtgga ggttca 36 28 16 DNA
Artificial Sequence A primer. 28 ggaggtggag gttctc 16 29 23 DNA
Artificial Sequence A primer. 29 tctgcggatc ccctagggac caa 23 30 36
DNA Artificial Sequence A primer. 30 aggtattgac tgaagtttag
cgagtgaggc attggt 36 31 36 DNA Artificial Sequence A primer. 31
ccacctgaac cgcctcctcc ggtaccagta taccag 36 32 30 DNA Artificial
Sequence A primer. 32 ccgggagaac ctccacctcc tgaacctcca 30 33 20 DNA
Artificial Sequence A primer. 33 accacggtct cgattacggc 20 34 20 DNA
Artificial Sequence A primer. 34 actactgctg aacggcgtcg 20 35 32 DNA
Artificial Sequence A primer. 35 cacacaggat ccgccgccga ggccggcatc
ac 32 36 32 DNA Artificial Sequence A primer. 36 cacacaggta
ccctgctgaa cggcgtcgag cg 32
* * * * *